Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.
Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Shahzad A
SA
Pandith
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Niha
N
Dhar
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Satiander
0000-0001-9197-8378
S
Rana
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Wajid Waheed
WW
Bhat
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Manoj
M
Kushwaha
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Ajai P
AP
Gupta
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Manzoor A
MA
Shah
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.).
Ram
R
Vishwakarma
Plant Biotechnology Division (S.A.P., N.D., S.R., W.W.B., S.K.L.), Quality Control and Quality Assurance Division (M.K., A.P.G.), and Medicinal Chemistry Division (R.V.), CSIR-Indian Institute of Integrative Medicine, Jammu Tawi 180001, India; andDepartment of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India (M.A.S.) sklattoo@iiim.ac.in.
Surrinder K
0000-0002-8902-3713
SK
Lattoo
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003198:Computer Simulation; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000096:biosynthesis; D018506:Gene Expression Regulation, Plant; D014644:Genetic Variation; D018745:Genome, Plant; D007700:Kinetics; D055442:Metabolome; D010802:Phylogeny; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012250:Rheum / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D053719:Tandem Mass Spectrometry
D000880:Anthraquinones; D018076:DNA, Complementary; D005419:Flavonoids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D048630:Polyketide Synthases