Introduction

Anthocyanins are water-soluble flavonoids with antioxidant properties responsible for coloration of flowers, seeds, and fruit in plants (Davies et al. 2012; Muñoz-Gómez et al. 2021; Jideani et al. 2021). Plant vegetative parts and trichomes accumulate anthocyanins, providing tolerance against biotic stresses such as pathogen attack and insect herbivory, as well as abiotic stresses like high light intensity and temperature fluctuations (Misra et al. 2010; Redondo-Gómez et al. 2013; Chen et al. 2019; Saxena et al. 2023a, b). Proanthocyanidins (PAs) are polyphenolic compounds abundant in plants. They are found in seeds, fruits, bark, leaves, and stems, contributing to the color, taste, and astringency of various plant-based foods and drinks (Samanta et al. 2011; Dasiman et al. 2022; Qi et al. 2023). PAs are integral to plant defense mechanisms against stresses such as pathogens and UV radiation (Hectors et al. 2012). They also play a role in seed coat formation and provide potential health benefits to both humans and animals (Bogs et al. 2005; Lattanzio et al. 2009; Mellway et al. 2009; Huang et al. 2010; Dixon et al. 2020). The flavonoid biosynthesis pathway consists of biosynthetic genes: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), and leucoanthocyanidin reductase (LAR) that synthesize anthocyanins and PAs from dihydro flavonols (Pandey et al. 2016; Naik et al. 2024). Anthocyanins are temporally and spatially accumulated in different parts of the plants, and their biosynthesis is regulated by the heterotrimeric transcription factors MBW complex, composed primarily of R2R3-MYB, bHLH, and WD40 proteins (Carey et al. 2004; Li et al. 2016; Naik et al. 2021). MYB regulatory proteins consist of a DNA-binding domain at the N-terminal, while the C-terminal is responsible for interaction with other proteins (Ambawat et al. 2013). The MYB domain consists of three α-helices, each 50–60 amino acid-long imperfect repeats categorized as R1, R2, and R3 MYBs (Rajput et al. 2022b).

These R2R3-MYB TFs are plant-specific regulatory proteins that regulate various processes such as specialized metabolism, abiotic stress responses, defense, phytohormone signaling, root genesis, growth, and development in plants (Dixon and Paiva 1995; Dubos et al. 2010; Naik et al. 2022). The MBW complex is crucial for regulating anthocyanin biosynthesis across various plant species (Ramsay and Glover et al. 2005). Expression of these proteins from different species in Arabidopsis mutants with reduced anthocyanin content has successfully restored anthocyanin levels. In A. thaliana, regulation of the flavonoid biosynthesis pathway is well understood. AtMYB75 (PAP1), AtMYB90 (PAP2), AtMYB113 (PAP3), and AtMYB114 (PAP4) regulate anthocyanin biosynthesis in the model plant Arabidopsis thaliana (Borevitz et al. 2000; Zimmermann et al. 2004; Zuluaga et al. 2008; Bac-Molenaar et al. 2015). Despite extensive research on flavonoids in crops such as Medicago, apple, peach, and tomato over the years, the regulatory mechanisms in chickpea has still not been fully elucidated (Li et al. 2012; Pang et al. 2007; Tohge et al. 2017; Rajput et al. 2022b; Saxena et al. 2023a, b). Peel et al. (2009) identified and characterized an R2R3-MYB (MtLAP1) that regulates anthocyanin in Medicago truncatula. In another study, the interaction of MtTT8 with MtWD40-1, and MtPAR or MtLAP1 results in MBW complexes, which in turn activates MtANS and MtANR promoters for triggering anthocyanin and PA accumulation, respectively (Li et al. 2016). In Cammelia sinensis, the overexpression of CsWD40 in A. thaliana transparent testa glabra 1 (ttg1) restored the normal trichome and seed coat developmental patterns (Liu et al. 2018). Co-expression of CsWD40 and CsMYB5e in tobacco plants led to the accumulation of anthocyanins and PAs content. VvMYBA1 and VvMYBA2 are key regulators of anthocyanin biosynthesis in grape berries (Rinaldo et al. 2015). Also, the orthologs of these proteins, MdMYBA/MdMYB1/MdMYB10 and PcMYB10, regulate anthocyanin biosynthesis in apple and pear (Pyrus communis), respectively. MdMYB10 regulates anthocyanin accumulation in the fruit flesh and affects the skin color across many apple genotypes (Li et al. 2012; Hu et al. 2016). Although MdMYBA and MdMYB1 are expressed in the red-colored fruit skin in a few genotypes, a recent study unveils that MdMYB110a, a paralog of MdMYB10, also regulates anthocyanin accumulation in the flesh of apple (Fang et al. 2023). In the Dendrobium hybrid, DhbHLH and DhMYB interact to regulate spatial and temporal anthocyanin accumulation in floral tissues (Wang et al. 2022). This interaction ensures that anthocyanin pigments are precisely localized and expressed at specific developmental stages, contributing to the vibrant coloration of the flowers. In addition to the role of MYB TFs activating the anthocyanin and PA biosynthesis, they can also act as a repressor in many cases, such as MaMYBPR in banana, FaMYB1 in strawberries, and VvMYB4 in grapevines (Paolocci et al. 2011; Pérez-Daz et al. 2016; Rajput et al. 2022a, b). The MBW module consisting of FaMYB5, FaEGL3, and FaLWD1-like directly targets the promoters of F3′H and LAR, thereby regulating the content of anthocyanins and PAs (Yue et al. 2023). Through this coordinated action, the MBW complex ensures the precise modulation of these secondary metabolites for optimal plant health and productivity. APETALA2 (AP2) transcription factors (TFs) disrupt the formation of the MBW complex, resulting in the repression of PA biosynthesis.

Chickpea (Cicer arietinum L.) is an annual legume crop and the second-most cultivated pulse crop in terms of production and consumption (Koul et al. 2022). Cultivated chickpeas are of two types according to seed and flower morphology: Kabuli and Desi. The seed coat color is an important agronomic trait due to enriched flavonoid and other antioxidant dietary components in chickpea (Pal et al. 2023). The physical appearance of Kabuli seeds, such as light color and large size, has long been a trait of consumer preference and trade value, in addition to being an important quality target component and adaptation trait (Bajaj et al. 2015). Desi seeds are darker in color due to anthocyanin and PAs accumulation in the seed coat (Rajput et al. 2022b; Pal et al. 2023).

This study characterizes two anthocyanin-specific SG6 MYB TFs, CaLAP1, and CaLAP2 in chickpea. They interact with the TT8-like CabHLH1, forming a trimeric complex with CaTTG1 (WD40), potentially activating anthocyanin and PA biosynthesis genes. Further, CaLAP1 and CaLAP2 partially restored anthocyanin deficiency in Arabidopsis mutants and when overexpressed in chickpea, increased the  accumulation of anthocyanin and PA in chickpea seed coats. These findings provide insight into anthocyanin and PA regulation in chickpea, offering the potential for developing varieties with higher anthocyanin and PA levels and improved stress tolerance.

Materials and methods

Plant material

Chickpea (Cicer arietinum L.) cultivars ICC4958 (Desi type) and ICCV2 (Kabuli type) plants were grown in the fields of the National Institute of Plant Genome Research (NIPGR), New Delhi, India. For genetic transformation, ICC4958 cultivar was used and grown in the plant growth chamber (Conviron, Winnipeg, Canada) at 22–24 °C with 60% humidity and a 10-h light period with an intensity of 250 μmol m−2 s−1. Crossing a triple myb mutant myb12, myb111, and myb11 (Stracke et al. 2007) with another triple myb mutant myb75, myb90, and myb114 (Appelhagen et al. 2011) produced an myb sextuple mutant. This mutant was detected in the F2 generation through PCR analysis and studied for metabolite accumulation. However, seedlings of this mutant failed to accumulate flavonols or anthocyanins (Naik et al. 2021). The wild type (Col 0), myb sextuple mutants, and the corresponding CaLAP-overexpressing lines of A. thaliana were grown in the plant growth chamber (AR-41L3; Percival, Perry, IA, USA) with a photoperiod of 16-h light/8-h dark at 22 °C. Nicotiana benthamiana plants were grown in a plant growth chamber with an 8-h light/16-h dark photoperiod at 22 °C. The A. thaliana suspension cell culture At7 was derived from the hypocotyl of the reference accession Columbia (Col) and handled as described in Stracke et al. (2016).

Identification of the anthocyanin-specific transcriptional regulators from C. arietinum

The functionally characterized anthocyanin-specific R2R3-MYB transcriptional regulators from various plants were subjected to a tblastn search against the C. arietinum genome sequence for the identification of candidate anthocyanin-specific R2R3-MYB regulators in chickpea. The corresponding protein sequence of CaMYB was aligned with the previously characterized landmark MYBs (Rajput et al. 2022b) via MAFFT v.7.299b (Katoh and Standley 2013). The alignment was trimmed via phyx and finally subjected to MEGA X (Kumar et al. 2018) for the construction of a neighbor-joining phylogenetic tree with a 1000 bootstrap value.

Gene expression analysis

C. arietinum RNA-Seq data sets for 10-day-old samples (whole seedlings, seedling roots, seedling shoots, shoot apical meristem, developmental stages of seeds, germinating seedling, developmental stages of flower, reproductive plant, root, vegetative plants and young leaves) (BioProjects: PRJNA182724, PRJNA79731, PRJNA316844, PRJNA316845, SAMN00794551) (Garg et al. 2011; Rajkumar et al. 2020) were retrieved from the Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra). STAR v2.5.1b (Dobin et al. 2013) was applied to align the reads to the C. arietinum genome sequence in the two-pass mode. Reads were mapped if the alignment similarity exceeded 95% and covered 90% of the read length as previously described (Haak et al. 2018). Feature Counts v1.5.0-p3 (Liao et al. 2014) was deployed with default settings to quantify gene expressions. The resulting count tables were processed and combined by previously developed Python scripts (Haak et al. 2018). Heatmap construction was performed with HCE (Hierarchical Clustering Explorer 3.5), and the hierarchical clustering of genes was executed by the Euclidean distance method (Seo et al. 2006).

Total RNA from various tissues of chickpea was isolated from GSure plant RNA isolation kit (GCC Biotech, Kolkata, India). The isolated RNA was treated with RNAse-free DNAse (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The RTq-PCR analysis of selected genes was conducted using a 2 × PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Each PCR mix contained 1 μl of diluted cDNA (equivalent to 25 ng total RNA), 5 μl of 2 × SYBR Green PCR Master Mix (Applied Biosystems), and 10 nM of each gene-specific primer in a final volume of 10 μl. All qRT-PCRs were done under the following conditions: 20 s at 95 °C, 3 s at 95 °C, and 40 cycles of 30 s at 60 °C in 384-well optical reaction plates (Applied Biosystems). The expression levels of various genes involved in anthocyanin and PA biosynthesis were assessed using the 7500 Fast Real-Time PCR System (Applied Biosystems). The integrity of the amplicons was confirmed by melting curve analysis conducted from 60 to 95 °C after 40 cycles. For normalization of transcript level, the housekeeping genes, chickpea elongation factor 1-α (EF-1α) (GenBank: AJ004960.1) and Caβ-tubulin (Caβ-Tub) (LOC101495306) coding sequence, were used. The cycle threshold (Ct) 2−ΔΔCT method was used to calculate the relation fold change (Livak and Schmittgen 2001). Three biological replicates and three technical replicates were used for mean value calculation. Primers used in the study are mentioned in Table S1.

Cloning of the transcription regulators from cDNA

The first-strand cDNA of C. arietinum cultivar ICC4958 was used as a template for amplifying the coding sequence of CaLAP1, CaLAP2, CabHLH1, and CaTTG1. The C. arietinum genome database sequence information was used to design  the primers. The Gateway™ attB-site-containing primers and Gateway™ cloning vector pDONRTMZeo (Invitrogen) were utilized for the prepration of entry clones. All entry clones were subsequently confirmed by Sanger sequencing at the sequencing core facility of NIPGR (New Delhi, India).

Subcellular localization of CaLAPs

The coding sequence of CaLAP1 and CaLAP2 were recombined to CaMV35S promoter-driven C-terminal YFP in PGWB441 binary vector using Gateway™ LR-recombinase (Nakagawa et al. 2007).The resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101::pMP90 (Koncz and Schell 1986). The cultures and nuclear marker (NLS–RFP) were mixed with freshly prepared infiltration buffer (10 mM MES-KOH, pH 5.7, 10 mM MgCl2, 150 μM acetosyringone) followed by co-infiltration in the leaves of 3-week-old plants of N. benthamiana and kept at 22 °C for 48 h. The infiltrated leaves were observed under a Leica TCS SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany). RFP fluorescence was detected with excitation at 558 nm and emission at 583 nm, while YFP fluorescence was observed with excitation at 513 nm and emission at 530 nm.

Yeast two-hybrid (Y2H) assay

The entry clones of CaLAP1 and CaLAP2 were recombined in prey vector pGADT7g (Clontech Laboratories Inc., San Jose, CA, USA), and CabHLH1, CaTTG1 in the bait vector pGBKT7g (Clontech Laboratories Inc.). The Y2H Gold strain was initially cultured in yeast extract–peptone–dextrose (YPD) broth medium at 30 °C for 24 h. Following incubation, the culture was harvested by centrifugation at 3420 g for 5 min. The resulting pellet was then co-transformed by both prey and bait constructs plasmids into yeast gold strain using the EZ-yeast transformation kit (MP Biomedicals, Irvine, CA, USA) spread in SD/-Leu/-Trp (DDO, Double Dropout), lacking medium. The pGADT7g-CaLAP1, pGADT7g-CaLAP2, and pGBKT7g-CabHLH1, pGBKT7g-CaTTG1 containing yeast colonies were patched in SD/-Leu/-Trp (DDO, double dropout), SD/-Trp/-Leu/-His/-Ade (QDO, quadruple dropout), and 5 mM 3-amino-1,2,4-triazole (3-AT), X-α-gal -QDO plates and grown at 30 °C for 3 days. pGADT7-large with T/pGBKT7–53, and pGADT7-Iarge T/pGBKT7-Lamin combinations were used as a positive and negative control (Pipas and Levine 2001). Three repetitions of the experiment were performed, ensuring the inclusion of both positive and negative controls. Consistency in results was observed throughout.

Bimolecular fluorescence complementation (BiFC) assay

For BiFC assays, the entry clones of CaLAP1 and CaLAP2 coding sequences were recombined through LR clonase into the CaMV35S-driven vectors pSITE–YFPN and CabHLH1, CaTTG1 into the pSITE–YFPC (Martin et al. 2009). The resulting plasmids were transformed individually into Agrobacterium tumefaciens strain GV3101:pMP90 (Koncz and Schell 1986). Agrobacterium culture of both YFPN and YFPC fusion proteins construct was dissolved in freshly prepared infiltration medium (10 mM MES/KOH, pH 5.7, 10 mM MgCl2, and 150 μM acetosyringone) and co-infiltrated (1:1 ratio) in leaves of 3-week-old N. benthamiana. The negative control set consisted of CaLAP1/2-pSITE–YFPN with EV-pSITE–YFPC and pSITE–YFPN, alongside CabHLH1/CaTTG1-pSITE–YFPC. After 48 h of incubation at 22 °C, YFP fluorescence (excited at 513 nm, emitted at 530 nm) was visualized using a Leica TCS SP5 laser-scanning confocal microscope at the NIPGR confocal facility. The experiment was repeated three times, with consistent positive and negative controls included each time. The results remained consistent across all three repetitions.

Luciferase complementation imaging (LCI) assay

For LCI, full-length CDS sequences of CaLAP1 and CaLAP2 entry clones were ligated in pCAMBIA1300-LUCN and CabHLH1 in pCAMBIA1300-LUCC (Chen et al. 2008). The resulting plasmids were transformed into Agrobacterium tumefaciens GV3101::pMP90 (Koncz and Schell 1986) and the cultures were individually suspended in infiltration medium (10 mM MES-KOH, pH 5.7, 10 mM MgCl2,10 mM and 150 μM acetosyringone) and co-infiltrated in equal volume into N. benthamiana leaves. Luciferase substrate 100 μM D-luciferin was spread in infiltered N. benthamiana leaves after 48 h. A low-light charged-coupled device (CCD) imaging apparatus (Bio-Rad, Hercules, CA, USA) was used to quantify luciferase activity. The experiment utilized four N. benthamiana plants for each construct combination, with three repetitions performed. Results remained consistent with all repetitions.

Complementation analysis in A. thaliana

The entry clones of CaLAP1 and CaLAP2 were recombined using LR clonase into the destination vector pLEELA harboring 2 × 35S CaMV promoter and BASTA resistance for the selection. Resulting plasmids were transformed into A. tumefaciens strain GV3101::pM90RK (Koncz and Schell 1986) using electroporation. T-DNA insertion constructs containing CaLAP1 and CaLAP2 CDS were transformed into the myb sextuple (myb12, myb111, myb11, myb75, myb90, myb114) mutants of A. thaliana via Agrobacterium-mediated floral dip method (Clough and Bent 1998). BASTA-resistant A. thaliana seedlings were transferred to the soil-filled pots for further analysis.

Co-transfection analysis in A. thaliana At7 protoplasts

The co-transfection assay was performed using A. thaliana At7 protoplasts. The coding sequences of CaLAP1, CaLAP2, CabHLH1, and CaTTG1 were incorporated into the Gateway-compatible pBT-Dest destination vector, under the control of a 2 × 35S promoter, to generate the effector construct (Baudry et al. 2004). For the reporter constructs, entry clones having promoter sequences of CaCHS6 and CaDFR2 were recombined in the pDISCO destination vector. The cultivation of At7 cells, co-transfection of effector and reporter constructs into protoplasts, and the determination of activation capacity followed the methodology outlined in Stracke et al. (2010, 2016), Rajput et al. (2022b) and Saxena et al. (2023a, b).

Yeast one-hybrid (Y1H) assay

The Y1H assay was performed using the Y1H gold yeast strain (Takara Bio Inc., Shiga, Japan). The promoter fragments of CaCHS (1141 bp), CaDFR2 (735 bp), CaANS (869 bp), and CaANR (1325 bp) were cloned into the pABAi vector (Cat. No. 630491, Takara Bio Inc.). The pABAi vector harboring promoter fragments was linearized with Bsp119I (BstBI) enzyme and transformed into Y1H Gold strain according to the Matchmaker™ manual. The CaLAP1 and CaLAP2 CDS fragments were also cloned into the pGADT7-gateway vector. The construct plasmids having the desired genes and empty pGADT7-gateway vector as control were transformed into pre-transformed Y1H strains containing target promoter sequence. The interactions were checked on Aureobasidin (AbA)-supplemented media. The growth of different promoter-integrated Y1H strains was checked under –Leu/SD media having different concentrations of AbA to determine the MIC (minimum inhibitory concentration) values.

Electrophoretic mobility shift assay (EMSA)

The electrophoretic mobility shift assay (EMSA) was performed using probes with biotin-labeled dUTP and the Light-Shift Chemiluminescent EMSA Kit (Thermo Scientific) as described previously (Xu et al. 2017). DNA fragments of CaDFR2 promoter (53 bp) and CaANS promoter (48 bp) (Table S1) containing the CaLAP1/2-binding sequence were used. Both sense and antisense for the regular (WT) and mutated (MT) oligonucleotides were biotin labeled as described in the manual (biotin 3’ end labeling kit). For bacterial protein expression, the specific primers (supplementary text Table S1) were used to amplify the CaLAP1-CDS and CaLAP2-CDS by using the clones of CaLAP1 and CaLAP2, respectively, and ligated at BamHI and HindIII restriction sites in pET28a + protein expression vector to express His-tagged proteins. His-tagged recombinant proteins expressed E coli BL21 (codon +) using the 0.5 mM IPTG and purified through Ni–NTA-beads.

Binding reaction for EMSA was set as described in the manual. Briefly, 1 µg His-tagged recombinant proteins containing 1 × binding buffer were incubated together with biotin-labeled probes in 20 µl reaction mixtures (containing 10 mM Tris–HCl, 150 mM KCl, 1 mM DTT, 50 ng/ml poly (dI-dC), 2.5% glycerol, 0.05% Nonidet P-40, 100 mM ZnCl2, and 0.5 µg/ml BSA) for 45 min at 25 °C and separated on 8% native polyacrylamide gels in 1 × TAE (Tris–acetate EDTA) buffer. The labeled probes were detected by using streptavidin–horse radish peroxidase (HRP) and chemiluminescence reagents according to the instructions provided with the EMSA kit. The figure legends provide information on the amount of protein (1 µg) for binding with the biotin-labeled probe (2 nM).

Dual-luciferase assay

The entry clones of proCaANS (1930 bp) and proCaANR (1816 bp) were recombined into the p635nRRF containing 35S:REN vector while entry clones of CaLAP1, CaLAP2, CabHLH1, and CaTTG1 were recombined into the PGWB420 destination vector (Ezquerro et al. 2023). These reporter and effector constructs were co-infiltered in different combinations in N. benthamiana leaves to determine the transactivation activity. Using the dual-luciferase reporter assay system (Promega, Madison, WI, USA), firefly luciferase and REN activity were assessed in the extracts of leaf discs after 48 h of co-infiltration, following the manufacturer's instructions. With the help of the POLAR star Omega multimode plate reader (BMG Labtech, Ortenberg, Germany), the samples were quantified, and the firefly luciferase activity was normalized to that of the REN. Four leaves of two plants were utilized for each combination of constructs, and the experiment was repeated twice. Consistency in results was observed across both repetitions.

Generation of transgenic chickpea lines

Transgenic chickpea lines were developed as described previously (Khandal et al. 2020; Pal et al. 2023; Saxena et al. 2023a, b). Briefly, chickpea (ICC4958) seeds were surface sterilized with ethanol and 2% sodium hypochlorite, followed by soaking for about 7–8 h. A healthy mature seed's half cotyledon and embryo were used as explant for agro-transformation of the construct of interest. The explant was incubated with the agro-suspended culture containing half-strength Murashige and Skoog (MS) medium (pH 5.5) and 100 μM acetosyringone for 30 min. The transformed explants were shifted to the half-strength MS plates for 48 h in the dark at 22 °C. Subculturing of these explants having radicle was done on the plates containing the shoot and root induction half-strength MS medium (pH 5.8) containing cefotaxime (250 mg/l) and kanamycin (200 mg/l). The healthy transformants were micro-grafted on the 7- to 10-day-old root stocks from the same accession. The acclimatization of these micro-grafted plants was done on a growth chamber (Conviron, Winnipeg, Canada) at 22–24 °C with 60% humidity and a 10-h light period with an intensity of 250 μmol m−2 s−1 to maturity.

Total anthocyanin quantification

Total anthocyanin content was quantified using the spectrophotometric method as described previously (Pandey et al. 2014, 2015). Briefly, 2.5 ml of acidic methanol (1%, v/v) was added to 0.25 gm of lyophilized fine ground tissue, and the extract was incubated overnight at 4ºC in the dark. The mixture-lysate was centrifuged at 3000 g, the supernatant was collected, and absorbance was measured at 530 and 657 nm. The anthocyanins were quantified using the formula: (A530–0.25 × A657)/tissue weight.

Total PA quantification

The seed coat of wild-type (ICC4958) and CaLAP1-2-OE lines was used for the extraction and quantification of soluble and insoluble PA as mentioned in Pang et al. (2007). For estimating the PA content in samples, different dilutions of catechin were used to develop the standard calibration curve (200, 400, 600, 800, and 1000 µg/ml).

Lyophilized seed coat samples were powdered and added with 1 ml of extraction buffer (butanol–HCl reagent (5:95, v/v) containing 0.7 g FeCl3/l) and vortexed, followed by sonication. The sonicated extract was filtered with 0.22 µm PVDF filters (Merck Millipore, Darmstadt, Germany). The absorbance was measured at 550 nm following a 1 h boil to determine the insoluble PA content. Samples were cooled to room temperature, and the absorbance was measured again at 550 nm. A standard curve of procyanidin B1 (Merck) at dilutions of 50, 100, 150, 200, and 250 µg/ml was developed to estimate PA content in the samples.

UHPLC and LCMS analysis of chickpea seed tissue

Monomeric and oligomeric PAs were estimated in three biological and three technical replicates, following the methodology described previously (Rajput et al. 2022b; Pal et al. 2023).. Lyophilized seed coat and embryo tissue samples (200 mg) were extracted with 80% methanol. The clear supernatant extract was filtered through 0.22 µm for analysis. Three volumes of 2 M acidic methanol:HCl were added and incubated for 45 min at 90 °C. The samples were dried in rotavapor and resuspended in 1 mL 80% methanol. The analysis was performed using a 1290 Infinity II series UHPLC system (Agilent 290 Technologies, Santa Clara, CA, USA) enabled with a Zorbax Eclipse Plus C18 column kept at 30 °C. The mobile phase composition was solution A (0.1% formic acid in HPLC-grade water) and solution B (0.1% formic acid in acetonitrile). However, the chromatographic parameters included a constant flow of 270 µl/min (injection volume, 3 µl) and a run time of 47 min, including equilibration time. Before analysis, all samples were sterilized using a 0.22 µm PVDF syringe filter (Merck). For the anthocyanin quantification, we used the LC–MS systems. A UHPLC system (Exion LC Sciex, Framingham, MA, USA) connected to a triple quadrupole (QTRAP6500 +) (ABSciex, Framingham, MA, USA) using electrospray ionization was used to analyze the PAs. The positive ionization voltage was set at 5500 V. Utilizing the Analyst software (version 1.5.2), the mass spectrometer was used in a variety of reaction monitoring modes for both qualitative and quantitative analysis. Analytical standards (Merck) were used for each compound.

Statistical significance

We utilized one-way ANOVA followed by Tukey's post hoc test to compare multiple datasets (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001). Each dataset comprised three replicates.

Results

Identification of anthocyanin-specific R2R3-MYB regulators from C. arietinum

In our previous study, an in silico analysis led to the identification of 119 putative R2R3-MYB regulators in chickpea (Rajput et al. 2022b). Here, we identified two homologs of R2R3-MYB activators, CaLAP1 (CaMYB16) and CaLAP2 (CaMYB30), exhibiting high similarity to the known anthocyanin regulators associated with legume anthocyanin production (LAP). CaLAP1 and CaLAP2 encode the proteins comprising 250 and 236 amino acids, respectively. The phylogenetic study showed the clustering of CaLAP1 and CaLAP2 with the previously characterized anthocyanin-specific R2R3-MYBs (Fig. 1a). The amino acid alignment revealed that CaLAP proteins share a highly conserved N-terminal R2R3-MYB domain having COP1-interacting VP motif (VP [E/D] RAG), bHLH-interacting consensus motif ([D/E] Lx2[R/K]x3Lx6Lx3R) and dicot-specific conserved ANDV motif that distinguishes it from the monocot-specific DNEI motif. Additionally, the C-terminal region contains an anthocyanin-specific SG6 motif (KPRPR [S/T] F) (Fig. 1b).

Fig. 1
figure 1

Phylogenetic analysis, amino acid sequence alignment, and sub-cellular localization of candidate anthocyanin-specific R2R3MYB proteins in chickpea genome. a The depicted R2R3-MYB proteins were classified into three major groups: flavanol, anthocyanin, and proanthocyanidin biosynthesis-specific regulators using landmark flavonoid-specific R2R3-MYBs from different plant species. The phylogenetic tree was constructed using MEGA X. CaLAP1 and CaLAP2 are highlighted by purple ovals. b The R2 and R3 repeat within the MYB domain, alongside the bHLH interaction motif and the S6 defining motif, are annotated based on known anthocyanin-specific R2R3-MYB regulators. The VP domain, representing the COP1-interacting domain, is identified within the MYB R2 domain. c Subcellular localization of CaLAP–YFP fusion proteins in Agrobacterium-infiltrated tobacco leaves analyzed by confocal microscopy. NLS–RFP was used as a nuclear marker

Full size image

CaLAP proteins are nuclear localized

The subcellular localization of CaLAP proteins was confirmed by transient expression of gene constructs encoding CaLAP proteins fused to yellow fluorescent protein (YFP) and a nuclear marker red fluorescence protein (RFP) fused to a nuclear localization signal (NLS–RFP) in N. benthamiana leaves. CaLAP1–YFP and CaLAP2–YFP fusion proteins were expressed exclusively in the nucleus and co-localized with the nuclear marker NLS–RFP suggesting that CaLAP1 and CaLAP2 are nuclear proteins (Fig. 1c).

The expression patterns of CaLAP correlate with putative structural target gene expression

We conducted an analysis using a publicly available RNA-Seq dataset comprising different tissues and developmental stages of seeds and flowers to analyze the expression of CaLAP1 and CaLAP2, along with CabHLH1 and CaTTG1 involved in the MBW complex formation and anthocyanin and PA biosynthesis enzymes coding genes. CaLAP1 expression was abundant in root and early developmental stages of seeds (S1–S4) and found in varying levels in different stages of flowers, whereas a very low expression of CaLAP2 was observed in the early developmental stages of seeds (S1–S4). The expression levels of CaANR and CaLAR were notably elevated during the seed developmental stages, particularly at stage 6 and stage 7, while showing varying expression levels in other tissues. CabHLH1 expression was higher at all stages of seed development and flowers, while CaTTG1 expression was constitutive and abundant in all tissues. Likewise, the expression of the putative anthocyanin biosynthesis genes, CaDFR2 and CaANS, showed a strong correlation with regulatory gene expression (Fig. 2a). The RT-qPCR data revealed a significantly higher expression of CaLAP1, CaDFR2, and CaANS genes in flower tissue as compared to seed coat and cotyledon in the Desi type. Conversely, CaLAP2 expression was notably elevated in the Kabuli type cultivar (Fig. 2b). A robust correlation was identified between the expression levels of the candidate CaLAP1 genes and CaANS, CaDFR2, CaANR, and CaLAR, particularly with higher expression observed in the seed coat. In contrast, CaLAP2 exhibited lower expression in the seed coat despite overlapping expression patterns. These findings suggest that the R2R3-MYB (CaLAP1 and CaLAP2) may function as positive regulators of anthocyanin biosynthesis in chickpea.

Fig. 2
figure 2

Expression analysis of regulatory and structural biosynthesis genes in two Cicer arietinum cultivars with different flower and seed coat colors. a Differential expressions of regulatory genes (CaLAP1, CaLAP2, CabHLH1, CaTTG1) and structural biosynthesis genes (CaDFR2, CaANS CaANR, CaLAR) were calculated from publicly available RNA-Seq data sets of various plant parts and developmental stages of flowers and seeds. b Expression of CaLAP, CaDFR2 and CaANS genes in flower and mature seed coat and embryo/cotyledon. The presented RT-qPCR data cover two independent biological replicates with three technical replicates of each and the error bars give ± SD values. CaEF-1α and CaβTub expressions were used as a reference control

Full size image

CaLAP1 and CaLAP2 partially restore the anthocyanin-deficient phenotype of the A. thaliana myb mutant

Anthocyanin-deficient myb sextuple mutant (Naik et al. 2021) was used to assess the in planta function of CaLAP1 and CaLAP2 as anthocyanin regulators under the control of the CaMV35S promoter. Transgenic seeds were grown on the half-strength MS medium supplemented with 4% sucrose. The accumulation of anthocyanin was observed in seedlings of Col 0 (wild-type control), which turned dark purple, whereas the myb mutant seedlings showed no anthocyanin accumulation. However, seedlings of transgenic myb mutant lines bearing the 35S::CaLAP1 and 35S::CaLAP2 constructs showed a light purple color, indicating partial accumulation of anthocyanin (Fig. S1). These results suggested that CaLAP1 and CaLAP2 have the potential to partially complement the anthocyanin deficiency phenotype of myb sextuple mutants of A. thaliana.

CaLAP1 and CaLAP2 interact with CabHLH1 and CaTTG1

Since CaLAP1 and CaLAP2 both contain a bHLH-binding motif ([DE]Lx2[RK]x3Lx6Lx3R) and anthocyanin biosynthesis depends on the formation of a regulatory MBW complex, we tested the potential interaction of these proteins with CabHLH1 and CaTTG1 co-factor. We selected our previously characterized chickpea bHLH1 protein (Rajput et al. 2022b; Pal et al. 2023) as a candidate to analyze the interaction with CaLAP1 and CaLAP2. The Y2H results indicate that both CaLAP1 and CaLAP2 interact with CabHLH1 (Fig. 3a). To validate the results in planta, we performed BiFC and LCI assay. In BiFC assay, reconstruction of YFP fluorescence was detected when CaLAP1–YFPN or CaLAP2–YFPN with CabHLH1–YFPC pairs were transiently expressed in N. benthamiana leaves (Fig. 3b). LCI assay further confirmed the interactions by showing luminescence signal between the CaLAP1–CabHLH1 and CaLAP2–CabHLH1 pairs (Fig. 3c). Since a functional MBW complex needed a WD40-repeat protein, we also tested the interaction of CaLAP1 and CaLAP2 with CaTTG1. Y2H results confirmed interactions between both CaLAP1 and CaLAP2 with CaTTG1. The in planta interaction was also validated through BiFC assays (Fig. S2). This supports the hypothesis that these proteins may play a significant role in anthocyanin regulation through participation in MBW complexes.

Fig. 3
figure 3

CaLAP1 and CaLAP2 interact with CabHLH1 as a part of the functional MBW complex. a Yeast two-hybrid (Y2H) assays showed in vivo interaction between CaLAP1 and CaLAP2 proteins with CabHLH1. AD, Gal4 activation domain; BD, Gal4 DNA-binding domain; DDO, double synthetic dropout (SD)-Leu-Trp medium; QDO/X, quadruple SD-Leu-Trp-Ade-His + X-α-Gal. b Bimolecular fluorescence complementation (BiFC) assay indicates planta interaction of CaLAP1, CaLAP2 with CabHLH1. Scale bar = 20 μm. c Luciferase complementation imaging (LCI) assay showing the interaction between CaLAP1 and CabHLH1 and CaLAP2 and CabHLH1

Full size image

CaLAP proteins form functional MBW complexes to activate anthocyanin and PA biosynthetic gene promoters

To establish connections between TFs and their targets, we conducted an analysis focusing on putative MYB-binding sites (MBS) within the promoters of genes involved in anthocyanin and PA biosynthesis. Initially, we examined the promoters of CaCHS6, CaDFR2, CaANS, and CaANR to identify potential MBSs. Structural analysis of the promoter regions, upstream of the transcription start site, identified several potential MBS motifs.To further extend the target specificities of CaLAP1 and CaLAP2 toward anthocyanin biosynthesis gene promoters, we performed PEG-mediated transient transfection in A. thaliana protoplasts. MBW complex-forming R2R3-MYB proteins CaLAP1, and CaLAP2, along with CabHLH1 and CaTTG1, were used in combination as effector constructs, while proCaCHS6 and proCaDFR2 were the reporter constructs. The co-transfection analyses suggested that CaLAP1 and CaLAP2 alone transactivate proCaCHS6 and proCaDFR2 to some extent, while in combination with CabHLH1 and CaTTG1 further enhanced the transactivation of proCaCHS6 and proCaDFR2 considerably (Fig. 4a). We also tested the potential of CaLAP1 and CaLAP2 in combination with AtEGL3 effector constructs in an At7 protoplast using proAtLDOX, proAtDFR, and proAtCHS reporter constructs that were assayed for their activity under the control of the CaMV35S promoter. We found the weak regulatory activity of CaLAP1 and CaLAP2 as compared to AtPAP1 effectors to activate anthocyanin biosynthesis in vivo in a heterologous system (Fig. S3).

Fig. 4
figure 4

CaLAP1 and CaLAP2 form an MYB–bHLH–WD40 (MBW) complex, which transactivates and binds to the promoters of genes involved in anthocyanin and proanthocyanin biosynthesis. a Results from co-transfection experiments in Arabidopsis protoplasts. Reporter constructs consisting of GUS driven by 1137-bp CaCHS6 and 877-bp CaDFR2 promoter fragments (reporter) were assayed for their transactivation by the 35S promoter-driven effectors CaLAP1, CaLAP2, CabHLH1, and CaTTG1, either alone or in the indicated combinations. Data are shown as the means of normalized GUS activity. b Yeast 1-hybrid assay. CaLAP1 and CaLAP2 physically interact with the promoters of CaCHS6, CaDFR2, CaANS, and CaANR. The physical interaction between CaLAP1, CaLAP2, and the proCaCHS6, proCaDFR2, proCaANS, and proCaANR was examined on leucine dropout media (-Leu) supplemented with an appropriate concentration of Aureobasidin (AbA). Strains transformed with the pGADT7 vector served as negative controls. The minimum inhibitory concentration (MIC) value for proCaCHS6 and proCaANR was determined to be 900 ng/ml AbA, while it was 800 ng/ml for proCaANS and 600 ng/ml for proCaDFR2. c Gel-shift assay (EMSA) using a 53-bp region of CaDFR2 promoter and 48-bp region of CaANS promoter surrounding the putative binding sequences and CaLAP1 and CaLAP2 proteins. Each lane has 5 nM of biotin-labeled DNA probe and protein with either mutated probe (MT) or wild-type (WT) probe and CaLAP1 or CaLAP2 protein with 1 µg concentration. d Dual-luciferase experiments were performed transiently in Nicotiana benthamiana leaves. Constructs harboring the firefly luciferase reporter gene (LUC) are driven by CaANS-1930 bp, and CaANR-1816 bp promoter fragments and were transiently co-infiltrated with CaLAP1, CaLAP2, CabHLH1, and CaTTG1 effector constructs, either alone or in the mentioned combinations. Data are presented of four biological and two technical replicates with ± SD

Full size image

To test the physical interactions between CaLAP1 and CaLAP2 with the promoters of CaCHS6, CaDFR2, CaANS, and CaANR, Y1H was conducted. proCaCHS6 and proCaDFR2 were found to harbor two MYB-binding sites each, while proCaANS possessed two MBS sites and one MYBCORE site, and proCaANR featured three MYB-binding sites (Fig. S4). Both CaLAP1 and CaLAP2 exhibited binding affinity toward proCHS6-1325, proCaDFR-735, proCaANS-869, and proCaANR-1141. Conversely, the respective empty vector controls did not display such interactions. These findings suggest that proCaCHS6, proCaDFR2, proCaANS, and proCaANR are probable target genes of both CaLAP1 and CaLAP2 (Fig. 4b). Additionally, to know about the direct binding of CaLAP1 and CaLAP2 transcription factors on proCaDFR2 and proCaANS, we performed gel-shift assay (EMSA). We found that the sequences TAACCT for proCaANS and TAACAG for proCaDFR2 are the sites at which CaLAP1 and CaLAP2 bind (Fig. 4c and Table S1).

Further, to assess the transactivation activity of the effectors on the promoters of CaANS and CaANR genes, dual-luciferase assays were performed in N. benthamiana leaves. Reporter constructs were co-infiltrated with the effector constructs in the following combinations: CaLAP1 and CaLAP2 individually, CaLAP1 and CaLAP2 separately with CabHLH1, and the combination with both CabHLH1 and CaTTG1. Interestingly, both reporter constructs, proCaANS and proCaANR, exhibited less transactivation activity with effectors CaLAP1 and CaLAP2 individually. However, the transactivation activity drastically increased when CabHLH1 was also present. The promoter of CaANS and CaANR showed the highest transactivation activity in the presence of all three effector constructs including CaLAP1 or CaLAP2, CabHLH1, and CaTTG1, suggesting a synergistic role in activating the promoters of CaANS and CaANR (Fig. 4d).

CaLAP1 and CaLAP2 modulate total anthocyanin and PA contents in chickpea seed coat

The chickpea transgenic lines overexpressing CaLAP1 and CaLAP2 under the control of CaMV35S promoter were found to be morphologically similar to the control plants except for the seed pigmentation, which was darker in the overexpressing lines (Fig. 5a). To assess the changes in the the metabolite content in CaLAP-overexpressing lines, we performed the spectrometric quantification of total anthocyanin and PA content (Fig. 5b and c). The analysis showed significantly higher anthocyanin and PA content in the seed coat of overexpressing lines than in the control lines suggesting that the overexpression of CaLAP1 and CaLAP2 enhances the biosynthesis of these pigments in chickpea.

Fig. 5
figure 5

CaLAP1 and CaLAP2 overexpression induces anthocyanin and PAs accumulation in the seed coat of C. arietinum. a Phenotype showing a clear distinction in the seed pigmentation due to overaccumulation of anthocyanins and PAs in the CaLAPs-OE lines of C. arietinum. b Representative visuals indicating the content of both soluble and insoluble PAs. c Total anthocyanincontent was determined using a spectrophotometer in both ICC4958 control chickpea lines and CaLAP1 and CaLAP2 overexpressing lines. d Soluble PAs were quantified using the DMACA reagent with catechin as the standard for absolute quantification. Insoluble PAs were estimated from dried seed tissue using the butanol–HCl reagent, with procyanidin B1 as the standard for absolute quantification in both control and transgenic seeds. The transgenic lines exhibited elevated levels of total anthocyanin accumulation and total PAs (soluble and insoluble). The values represent the means ± standard deviations of two biological replicates, with three technical replicates each for control and overexpressing lines

Full size image

CaLAP1 and CaLAP2 induce expression of the anthocyanin and PA biosynthetic genes in chickpea

Next, we performed gene expression analysis of regulatory and biosynthetic genes in the seedcoat of immature green seeds and the same embryo/cotyledon tissue of independent (L1, L2, and L3) chickpea CaLAP1-OE and CaLAP2-OE lines. In the overexpressing lines of CaLAP1, the CaLAP1 gene expression showed a 10-fold increase in the seed coat and 2–4-fold in the embryo/cotyledon, while CaLAP2 expression displayed up to 10- to 22-fold upregulation in seed coat tissue and 2–3-fold in embryo/cotyledon in the CaLAP2 overexpressing lines as compared to the control lines. The transcript levels of CaDFR and CaANS were elevated by 2- to 4-fold in the seed coat, while CaANR and CaLAR exhibited upregulation by 2- to 4- and 4- to 6-fold, respectively, in seed coat tissue compared to the control line. Similarly, CaLAR and CaANR also showed increased expression (Fig. 6a). Additionally, enhanced expression of CaDFR, CaANS, CaANR, and CaLAR was observed in embryo/cotyledon tissues of the same transgenic lines (Fig. 6b).

Fig. 6
figure 6

Expression analysis of anthocyanin regulators and biosynthetic genes in control and CaLAP1 and CaLAP2 overexpressing lines of seed coat and cotyledon tissue of C. arietinum. a Differential gene expressions of CaLAP1, CaLAP2, CaDFR1&2, CaANS, and CaANR show significantly higher transcript in CaLAP1 and CaLAP2-OE lines as compared to the ICC4958 control seed coat tissue. b Differential gene expressions of CaLAP1, CaLAP2, CaDFR1&2, CaANS, and CaANR show higher transcript values in a few lines of CaLAP1 and CaLAP2-OE lines as compared to ICC4958 control in embryo cotyledon tissue. The RT-qPCR data cover three technical triplicates, and the error bars give ± SD values. The expression level in control was set as 1 and relative expression levels are given. CaEF-1α expression was used as the reference control

Full size image

CaLAP1 and CaLAP2 modulate derivatives of anthocyanin and PA contents in the chickpea seed coat

To further substantiate our findings, we measured the anthocyanidin and PA content through LC–MS in the seed coat tissue of control and CaLAP1 and CaLAP2 overexpressing lines of chickpea. Three anthocyanidins, cyanidin, delphinidin, and petunidin, accumulated in higher amounts (up to 4-fold) in overexpressing lines as compared to the control seed coat tissues of chickpea (Fig. 7a). In addition, monomeric PAs such as catechin, epigallocatechin, epicatechin gallate, as well as oligomeric PAs procyanidin A1, B2 and C1 were found to be significantly increased in the overexpressing lines as compared to the control plants (Fig. 7b and c). These findings suggest the role of CaLAP1 and CaLAP2 in regulating pigment production in chickpea.

Fig. 7
figure 7

Targeted metabolite profiling for quantitative estimation of anthocyanins and PAs in the control and transgenic lines of C. arietinum. a Estimation of anthocyanin derivatives (cyanidin, delphinidin, petunidin) in the control (ICC4958) and CaLAP1 and CaAP2-OE lines. Anthocyanin derivatives in seed coat tissues of the control (ICC4958) and CaLAPs-OE lines by LC–MS analysis. b Monomeric forms of PAs: catechin, epigallocatechin, and epigallocatechin. c Oligomeric PAs: procyanidin A1 and procyanidin B2 and procyanidin C1. The monomeric and oligomeric forms of PAs were estimated using ultra-high performance liquid chromatography (UHPLC). The error bar shows ± SD values of three biological and three technical replicates. DW dry weight

Full size image

Discussion

Chickpea seeds are well known for their high contents of protein, antioxidants, and flavonoids, including anthocyanin and PA (Jameel et al. 2021; Rajput et al. 2022b; Grasso et al. 2022; Saxena et al. 2023a, b). Using a transgenic approach, targeting biosynthetic genes and regulatory proteins hold significant potential for enhancing the flavonoid content in chickpea. The flower and seed colors are among the earliest phenotypic traits studied in plant genetics and inheritance. Several anthocyanin derivatives, including cyanidin, malvidin, petunidin, pelargonidin, delphinidin, and peonidin, have been shown to aggregate in the chickpea seeds, conferring brown color to the seed coat in Desi chickpea (Pal et al. 2023). The rough texture and astringent flavor are attributed to the presence of PAs derivatives (catechin, epigallocatechin, and epicatechin gallate) in the chickpea seed coat (Soares et al. 2020). In our earlier investigation, it was observed that the RNAi knockdown lines of TT8-like CabHLH and multidrug and toxic compound extrusion (CaMATE) exhibited reduced levels of anthocyanin and PA derivatives in both flower and seed coats (Pal et al. 2023). Rajput et al. (2022b) characterized the PA-specific TFs, CaPAR1 and CaPAR2 TFs, providing insights into the complex regulation of PAs in chickpea.

The transcript levels of anthocyanin-associated biosynthesis genes, such as CaDFR and CaANS, which were notably elevated during flower and seed development stages, exhibited a significant correlation with the expression of CaLAP1, indicating its regulatory role in reproductive organs of the plants. A recent study conducted by Saxena et al. (2023a, b) indicates that CaANS exhibits elevated expression levels across various tissues, particularly within flower tissues. Conversely, CaLAP2 demonstrated low expression levels during the flowering stages, with limited expression observed in the early stages of seed development. An earlier study by Nesi et al. (2001) shows that the TT2 expression was confined to the seed, overlapping with BAN (BANYULS) expression and PA deposition patterns. In the presence of a functional TT8 protein in Arabidopsis, the gain-of-function mutant TT2 could induce ectopic expression of BAN in young seedlings and roots in Arabidopsis.

R2R3-MYB and bHLH TFs play crucial roles in anthocyanin and PA accumulation. The MYB–bHLH complex is responsible for changes in the color phenotypes in different tissues of plants (An et al. 2020; Sun et al. 2021). Anthocyanin content modulated through transcriptional regulatory proteins inspired us to characterize the anthocyanin-specific R2R3-MYBs proteins in chickpea. In our study, we found that both CaLAP1 and CaLAP2 interact with CabHLH1 and CaTTG1 TFs to form a trimeric complex in chickpea. This finding aligns with previous research, which has shown that members of the SG6-R2R3-MYB, bHLH, and WD40 repeats protein families regulate anthocyanin biosynthesis across various plant species (Lin-Wang et al. 2010; Xie et al. 2020). Our investigation in chickpea, a key pulse crop with significant global utilization, highlights its crucial role in the study of anthocyanin regulation and biosynthesis. In A. thaliana, PAP1 (MYB) and EGL3 (bHLH) interact to form the MBW complex with TTG1 to regulate the biosynthesis of anthocyanins, while TRANSPARENT TESTA2 (MYB) interacts with TT8 (bHLH) and TTG1 to regulate the biosynthesis of PA (Gonzalez et al. 2008; Li et al. 2020a, b). R2R3-MYB TFs harbor the conserved motif [D/E] LX2[R/K]X3LX6LX3R in the R3 domain, for the interaction with bHLH in addition to WD40 and form MBW complex that mediates anthocyanin biosynthesis (Kim et al. 2021). A similar kind of MBW regulatory module is known in different horticultural and flowering plant species where the SG6-R2R3-MYB TFs include Rosea1, Rosea2, and Venosa in Antirrhinum majus, MdMYB10 in apple, MYB75, MYB90, MYB113, and MYB114 in Arabidopsis, SlAN2-like in tomato, and VcMYBL1 in blueberry (Espley et al. 2007; Shang et al. 2011; Yan et. al 2020; Tang et al. 2021). Previous reports show that in Oenanthe javanica, OjMYB1 interacts with OjbHLH, and SmMYB75 interacts with TT8-like bHLH in eggplant which is responsible for regulating anthocyanin biosynthesis (Feng et al. 2018; Shi et al. 2021).

Co-transfection and dual-luciferase assays indicate that both CaLAP1 and CaLAP2 are independently capable of positively transactivating the activity of CaCHS6, CaDFR2, CaANS, and CaANR promoters. During co-infiltration with CabHLH1 and CaTTG1 proteins, the activity of the tested promoters significantly peaks, indicating the formation of a functional MBW complex. This suggests a synergistic interaction among these proteins in regulating gene expression. The heterologous expression of CaLAP1 and CaLAP2 in A. thaliana myb sextuple mutant suggests the partial restoration of anthocyanin in Arabidopsis seedlings. It might be due to the heterologous MBW complex formation from legume MYB and Arabidopsis EGL3 and TTG1 or due to the lower activity of MBW complex formed with a heterologous MYB protein on the promoters of Arabidopsis anthocyanin biosynthesis genes.

The transcript levels of anthocyanin-associated biosynthesis genes CaDFR2 and CaANS which were exclusively higher during the flower and seed development stages showed a strong correlation with CaLAP1 and CaLAP2 expression, suggesting the regulatory role of CaLAP1 and CaLAP2 in the reproductive organs. This data indicates increased levels of anthocyanins and PAs in seed coats, potentially contributing to protection against biotic stress (Ambawat et al. 2013). Indeed, the expression of CaLAP1 and CaLAP2 in flower and seed stages is higher and variable, which could be controlled by upstream activators or repressors transcriptionally and post-transcriptionally (Rajput et al. 2022a). Studies indicate that the accumulation of anthocyanins in chickpea plants is greatly influenced by the endogenous phytohormone content of the plant species (Brunetti et al. 2018 ; La Fountain et al. 2021). An et al. (2021) reported that the JAZ1 (JA-ZIM-domain)–TRB1(telomere-binding protein)–MYB9 module dynamically regulates the accumulation of anthocyanin and PA in response to jasmonic acid (JA) signaling. Auxin regulates the accumulation of anthocyanin and PA through the auxin response factor (ARF2), serving as a positive regulator of PAs, in A. thaliana (Jiang et al. 2022). The application of exogenous ethylene enhances anthocyanin accumulation in grape berries by promoting the transcription of key genes including CHS, flavanone-3-hydroxylase (F3H), leucocyanidin oxygenase (LDOX), and UDP-glucose:flavonoid-3-O-glycosyltransferase (UFGT) (El‐Kereamy et al. 2003).

Anthocyanins are essential for promoting plant reproduction, as their bright colors attract pollinators and seed dispersers (Petroni et al. 2011). Additionally, anthocyanins recurrently accumulate in young vegetative tissues and the sun-exposed tissues of fruits to guard against photoinhibition and photobleaching under light stress without significantly hampering photosynthesis (Gould et al. 2004). Light is a main environmental factor that affects anthocyanin levels, and HY5 controls the biosynthesis of anthocyanins by modulating the transcriptional activity of the MYB75/PAP1 TF in Arabidopsis, and B-box TF, MdBBX20 in apple, PpBBX18 and PpBBX21, associated with HY5 TFs in Pyrus pyriflora pear (Shin et al. 2013; Bai et al. 2019). Tomato hy5 mutants were generated using the CRISPR/Cas9 system to distinguish between HY5-dependent and -independent candidate TFs involved in anthocyanin biosynthesis (Qiu et al. 2019). In another study of the WRKY TF family, PyWRKY26 binds to the PyMYB114 promoter in red-skinned pears and increases its expression, acting as an anthocyanin regulator (Li et al. 2020a, b). Additionally, MdHY5–MdWRKY41–MdMYB regulatory module affects apple fruits' anthocyanin and PA synthesis (Mao et al. 2021). Moreover, miR828 and miR858 are studied to target the coding sequence of anthocyanin-specific MYB repressors in grapes (Tirumalai et al. 2019).

Seeds from CaLAP1 and CaLAP2 overexpressing lines exhibit a darker hue compared to control seeds, yet maintain their seed weight and size. A comparable study in Arabidopsis reported that the lighter seed color resulting from AP2 (APETALA2) overexpression indicates a reduction in PA content, highlighting AP2 involvement in repressing PA biosynthesis. AP2 binds directly to the promoter of MYBL2, thereby enhancing its expression. Furthermore, AP2's interaction with MYBL2 inhibits the formation of the MBW complex (Jiang et al. 2023), further underscoring its regulatory influence on PA biosynthesis and anthocyanin-related pathways. Other TFs like TTG2 (WRKY) rely on the MBW trimeric complex and contribute to the accumulation of PAs in the Arabidopsis seed coat. Although the anthocyanin content and transcript of CaDFR2 and CaANS are not well correlated in our results, it might be due to some post-transcriptional and post-translational changes occurring that might be modulating the anthocyanin content in the CaLAP1 and CaLAP2 overexpressing lines of chickpea. Jiang et al. (2020) reported that the MdMYB1 locus undergoes methylation because of apple Argonaute4 (MdAGO4s) binding to its promoter, thereby governing anthocyanin biosynthesis via the RNA-directed DNA methylation (RdDM) pathway.

The expression levels of anthocyanin and PA biosynthetic genes such as CaANS, CaDFR2, CaANR, and CaLAR are notably higher in the CaLAP1 and CaLAP2 overexpressing seed coats. Interestingly, some lines also exhibit higher expression in the embryo/cotyledon tissues. We observed that transgenic seeds exhibit a darker coloration compared to the wild type. Furthermore, the total anthocyanin and total PA content in these transgenic seeds are significantly higher, without adversely affecting seed size and weight. A similar kind of study reported in maize the function of PALE ALEURONE COLOR1 (PAC1) is essential, as it codes for a WD40 repeat protein closely resembling AN11 and TTG1. PAC1's involvement is vital for facilitating anthocyanin biosynthesis, particularly in the aleurone and scutellum of maize seeds. In our data. the variation in expression patterns suggests that seed coats may serve as storage tissues for anthocyanin and PA metabolites, serving as a buffer against various stresses and environmental challenges. Different forms of anthocyanins, including cyanidin, delphinidin, and petunidin, correlate well with the expression patterns of anthocyanin biosynthetic genes. Moreover, variations in anthocyanin content are observed within biological lines of CaLAP1 and CaLAP2 overexpression. Among the monomeric forms of PAs, such as catechin, epicatechin gallate, and epigallocatechin, higher accumulation is detected in transgenic lines, with catechin being a notably predominant PA, while the other two polymeric forms are of low level. This abundance of catechin provides robustness and protection against stress. Similar trends are observed in the accumulation of oligomeric forms of PAs in CaLAP1 and CaLAP2 overexpressing lines. This improvement in anthocyanin content, combined with elevated levels of PA, offers consumers access to seeds enriched with these beneficial compounds.

Moreover, higher levels of anthocyanins and proanthocyanins in transgenic seeds confer enhanced resistance to biotic and abiotic stresses. This attribute not only provides resistance in plants against various pathogens and environmental challenges, but also contributes to the overall health and resilience of the crop. The working model of the regulation of anthocyanin and PA in chickpea seed coats is also presented (Fig. 8). This model provides a comprehensive framework for genetically manipulating the anthocyanin production in chickpea to produce more nutritious crops enriched in anthocyanins and PAs. This can pave the way for future investigations into the role of R2R3-MYB TFs in different aspects of chickpea biology and crop improvement.

Fig. 8
figure 8

Working model for the genetic manipulation of anthocyanin and PA biosynthesis in chickpea. CaLAP1/2 regulates the transcription of anthocyanin and PA biosynthetic genes leading to high anthocyanin and PA accumulation in the overexpression lines of chickpea. High anthocyanin accumulation in the CaLAP1/2-OE lines causes darker seed coat color. MBS, MYB-binding sites; BBS bHLH-binding sites; CHS chalcone synthase; DFR dihydroflavonol reductase; ANS anthocyanidin synthase; ANR anthocyanidin reductase

Full size image