Abstract
Main conclusion
We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum.
Abstract
Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein–protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.
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Data availability
All data supporting the findings of this study are available within the paper and within the supplementary data published online.
Abbreviations
- ANR:
-
Anthocyanidin reductase
- BBS:
-
BHLH-binding sequences
- BiFC:
-
Bimolecular fluorescence complementation
- DMACA:
-
4-Dimethylaminocinnamaldehyde
- LAR:
-
Leucoanthocyanidin reductase
- MBS:
-
MYB-binding sequences
- PA:
-
Proanthocyanidin
- PAR:
-
Proanthocyanidin regulator
- TF:
-
Transcription factors
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Acknowledgements
This work was supported by the core grant of National Institute of Plant Genome Research and Department of Biotechnology grant (BT/PR36694/NNT/28/1722/2020) to AP. RR and JN acknowledge Council of Scientific and Industrial Research, Government of India, for Senior Research Fellowships. ST acknowledges Department of Biotechnology for Research Associate Fellowship. The authors are thankful to DBT-eLibrary Consortium (DeLCON) for providing access to e-resources. We acknowledge the Metabolome facility (BT/ INF/22/SP28268/2018) at NIPGR for phytochemical analysis.
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425_2022_3979_MOESM1_ESM.tif
Supplementary file1 Chromosomal distribution of CaMYB genes. The localization of 108 R2R3-MYB genes are present on all eight pseudochromosomes in the C. arientinum genome; however, 11 CaMYB genes, i.e., CaMYB109 to CaMYB119, could not be assigned to any pseudochromosome. The pseudochromosomes of C. arientinum are shown with vertical bars and their chromosome numbers are presented at the top. The segmentally duplicated gene pair is highlighted in gray and connected with a black dotted line, while the tandemly duplicated gene pair is shown with green color. The length of each chromosome is given in Mb at its bottom (TIF 8999 KB)
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Supplementary file2 The conserved sequence logo of R2 and R3 MYB repeats of the 119 CaMYB proteins. Black triangles represent the conserved tryptophan (W) residues in the R2R3-MYB domain. Amino acid residues involved in the formation of conserved helices are indicated by black horizontal lines at the bottom of the logos (TIF 7535 KB)
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Supplementary file3 Representative images of DMACA extracts of seed coats and cotyledons tissues for quantification of soluble and insoluble PAs in seed tissues of desi and kabuli types (PPTX 175 KB)
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Supplementary file4 Ultra-high-performance liquid chromatography (UHPLC) chromatograms of standards and different tissues of desi and kabuli types. STD- Different peaks in the chromatogram are of procyanidin B1 (1); epigallocatechin (2); catechin (3); procyanidin B2 (4); procyanidin C1 (5); procyanidin A1 (6) epicatechin gallate (7); CA1, seed coat of ICC4958; CA2, seed coat of BGD256; CA3, seed coat of ICC6253; CA4, seed coat of ICCV2; CA5, cotyledon of ICC4958; CA6, cotyledon of BGD256; CA7, cotyledon of ICC6253; CA8, cotyledon of ICCV2 (PPTX 213 KB)
425_2022_3979_MOESM5_ESM.pptx
Supplementary file5 Liquid chromatography–mass spectrometry (LC-MS) chromatograms of epigallocatechin (EGC); epicatechin gallate (ECG); catechin (C); procyanidin A1 (PC-A1); procyanidin B1 (PC-B1); procyanidin B2 (PC-B2); procyanidin C1 (PC-C1) (PPTX 107 KB)
425_2022_3979_MOESM6_ESM.pptx
Supplementary file6 Representative images of DMACA extracts of PA-deficient A. thaliana tt2-1 mutant, the corresponding Ler wild type and transgenic 35S::CaPAR1, 2 lines in tt2-1 background (PPTX 274 KB)
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Supplementary file7 Phylogeny of TTG1 proteins. C. arietinum TTG1-like proteins (Ca_XP004502764.1, Ca_XP004502765, and Ca_XP004493117.1) were employed for the construction of phylogeny using neighbor Joining (NJ) method at 1000 bootstrap replication along with TTG1 proteins from Ananas comosus (OAY82628.1), Anthurium amnicola (JAT63430.1), Arabidopsis lyrata (XP_020877598.1), A. thaliana (CAB45372.1), Arabis alpina (KFK27728.1), Arachis duranensis (XP_015944381.1), Brassica napus (ABP04013.1), Brassica oleracea (ADV03945.1), Brassica rapa (ADK11704.1), Cajanus cajan (KYP38612.1), Camelina sativa (XP_010493614.1), Carica papaya (XP_021909514.1), Cucumis melo (XP_008455615.1), Daucus carota (XP_017254238.1), Dichanthelium oligosanthes (OEL12645.1), Eucalyptus grandis (XP_010048445.1), Gossypium arboreum (XP_017612811.1), G. hirsutum (AAM95642.1), Helianthus annuus (XP_021969841.1), Jatropha curcas (XP_012089919.1), Lotus corniculatus (ARK19314.1), L. sessilifolius (ATI97635.1), Malus domestica (ADI58760.1), Medicago truncatula (AES72643.1), Momordica charantia (XP_022140838.1), Morus notabilis (XP_010099415.1), Nelumbo lutea (ALU11273.1), Nicotiana attenuata (OIT04713.1), N. sylvestris (XP_009782290.1), N. tabacum (ACJ06978.1), Noccaea caerulescens (JAU90849.1), Oryza sativa (BAS80310.1), Prunus avium (XP_021805417), P. persica (ACQ65867.1), Punica granatum (ADV40946.1), Rosa rugosa (AFY23208.1), Setaria italica (XP_004953461.1), Sorghum bicolor (XP_002452749.2), Solanum melongena (AJN91103.1), Spinacia oleracea (XP_021858376.1), Triticum urartu (EMS66145.1), Zea mays (NP_001310302.1). Four major clades are shown in different colors, where Ca_XP004502764.1 and Ca_XP004502765.1 (blue highlighted) were closely clustered with TTG1 proteins from other leguminous plants (L. corniculatus, L. sessilifolius, C. cajan, A. duranensis, and M. truncatula), while Ca_XP004493117.1 (red highlighted) was found in a separate cluster with the members of TTG1 proteins from poaceae family including D. oligosanthes, O. sativa, S. italica, S. bicolor, and T. urartu (TIF 2251 KB)
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Supplementary file8 Co-transfection experiment in A. thaliana protoplasts. Results from co-transfection experiments in A. thaliana protoplasts. A GUS-fused 1328 bp CaANR and 1289 bp CaLAR promoter fragment (reporter) was assayed for its responsiveness to various 35S promoter-driven effectors CaMYB16, CaMYB30, CaTT8 and CaTTG1, either alone or in combinations. The figure shows mean normalized GUS' activity resulting from the influence of tested effector proteins on proCaANR-1328 and proCaLAR-1289 (reporter). Data from a set of six replicates are presented (TIF 2572 KB)
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Rajput, R., Tyagi, S., Naik, J. et al. The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat. Planta 256, 67 (2022). https://doi.org/10.1007/s00425-022-03979-z
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DOI: https://doi.org/10.1007/s00425-022-03979-z