ABC Transporter-Mediated Transport of Glutathione Conjugates Enhances Seed Yield and Quality in Chickpea

Abstract

The identification of functionally relevant molecular tags is vital for genomics-assisted crop improvement and enhancement of seed yield, quality, and productivity in chickpea (Cicer arietinum). The simultaneous improvement of yield/productivity as well as quality traits often requires pyramiding of multiple genes, which remains a major hurdle given various associated epistatic and pleotropic effects. Unfortunately, no single gene that can improve yield/productivity along with quality and other desirable agromorphological traits is known, hampering the genetic enhancement of chickpea. Using a combinatorial genomics-assisted breeding and functional genomics strategy, this study identified natural alleles and haplotypes of an ABCC3-type transporter gene that regulates seed weight, an important domestication trait, by transcriptional regulation and modulation of the transport of glutathione conjugates in seeds of desi and kabuli chickpea. The superior allele/haplotype of this gene introgressed in desi and kabuli near-isogenic lines enhances the seed weight, yield, productivity, and multiple desirable plant architecture and seed-quality traits without compromising agronomic performance. These salient findings can expedite crop improvement endeavors and the development of nutritionally enriched high-yielding cultivars in chickpea.

Figure 1.

Frequency distribution curves and boxplots depicting the variation in multienvironment (years) field phenotyping data of three major pod/seed yield traits. A–F, PN (A, D), SN (B, E), and 100-SW (C, F), evaluated among the 291 desi and kabuli accessions (association panel) as a whole and accessions belonging to each desi and kabuli chickpea cultivar groups. The overall mean of phenotyping information across three environments were also used to generate frequency distribution curves and boxplots. Box edges signify the upper and lower quantiles, with the median value in the middle of the box. *P < 0.0001, two-sided Wilcoxon test.

Figure 2.

A–D, Determination of molecular diversity, relatedness, and historical recombination (LD) among all 291 desi and kabuli chickpea accessions (association panel) based on (A) unrooted phylogenetic tree construction, (B) population genetic structure determination, (C) PCA, and (D) LD decay (mean r²) estimation using 64,172 SNPs physically mapped on chromosomes. These molecular classifications, including population structure (at optimal population number K = 2, indicated by red and green colors), differentiated the 291 accessions into two major populations (POP I and POP II). B, In population structure, the accessions represented by vertical bars along the horizontal axis were categorized into K color segments according to their estimated membership fraction in each K cluster. C, In PCA, PC1, and PC2 explained 23.8 and 6.7% of the total variance, respectively. D, In LD decay, the plotted curved lines representing two populations (POP I and POP II) signify the mean r² values among SNPs spaced with uniform 50-kb physical intervals from 0 to 500 kb across chromosomes. The dotted lines denote the nonsignificant difference between expected and observed LD decay in the two populations.

Figure 3.

The SNP genotyping and phenotyping information of three major pod/seed yield component traits, PN, SN, and SW, evaluated among 291 desi and kabuli chickpea accessions were analyzed by environment (year) and using the overall mean across the three environments to generate Manhattan plots. The genomic distribution of SNPs mapped to all eight chromosomes of the kabuli genome, is indicated by the x axis. The y axis designates the −log₁₀ (P) value for SNP loci significantly associated with the studied traits. The SNPs exhibiting significant associations with the traits at a cutoff P value ≤ 10⁻⁴ are demarcated with dotted lines. SNPs significantly associated with SW at least in two environments (years) are indicated with black-dotted circles.

Figure 4.

Manhattan plots (A) depicting the significant P values of genomic SNP loci associated with 100-SW evaluated among the 291 desi and kabuli chickpea accessions. The genomic distribution of SNPs mapped on the eight chromosomes of the kabuli genome is indicated by the x axis. The y axis designates the −log₁₀ (P) value for SNP loci significantly associated with SW. The SNPs exhibiting significant association with SW at a cutoff P value ≤ 10⁻⁴ are demarcated with dotted lines. B and C, Local Manhattan plot (B) and high-resolution LD heat map (C) cover a 430-kb (35.10–35.53 Mb) genomic interval (highlighted with red dotted lines) surrounding a SW-associated significant SNP locus (C-SNP869[A/G]) on chromosome 2. Arrow specifies the genomic position of a trait-associated SNP on chromosome 2. In the LD heat map, r² indicates the frequency correlation between pairs of alleles across a pair of SNP loci.

Figure 5.

High-resolution molecular mapping and map-based cloning of a major QTL (CaqSW2.4) governing 100-SW in chickpea. A, Molecular mapping of a CaqSW2.4 QTL on chromosome 2 of two high-density intraspecific genetic linkage maps of desi (P_D, ICC 6013 × ICC 14649) and kabuli (P_K, ICC 13764 × ICC 19192) and on a consensus high-resolution genetic map (P_C) of chickpea. B, The integration of genetic and physical maps of the target genomic region (1.8 cM) harboring a CaqSW2.4 QTL mapped on a high-density consensus genetic map corresponded to a 437.7-kb sequence interval of chromosome 2. The 437.7-kb CaqSW2.4 QTL interval was further delimited to a 76-kb sequenced genomic region on chromosome 2 by integrating the aforesaid traditional QTL mapping with QTL region-specific association analysis in the 291 desi and kabuli accessions belonging to an association panel. C, Fine-mapping of a CaqSW2.4 QTL using two low- and two high-SW mapping populations of desi (380 F2 individuals of DLSW[ICC 6013-NIL]^CaqSW2.4 × DHSW[ICC 14649-NIL]^CaqSW2.4) and kabuli (277 F2 individuals of KLSW[ICC 13764-NIL]^CaqSW2.4 × KHSW[ICC 19192-NIL]^CaqSW2.4) scaled down from the 76-kb QTL interval to a 17.2-kb genomic region with two protein-coding genes, including an ABC transporter CaABCC3(6) gene, on chromosome 2. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW.

Figure 6.

Progeny testing-based individual phenotyping of six selected recombinants as well as low- and high-SW desi and kabuli NILs and mapping parental accessions to deduce the genotype of a delineated 17.2-kb CaqSW2.4 QTL governing SW in chickpea (A). Two protein-coding candidate genes annotated within a 17.2-kb CaqSW2.4 QTL genomic interval between the SNPs Ca235442357(A/C) and Ca235459613(T/C), of which the (C-SNP869[A/G]) synonymous SNP in the coding region of a CaABCC3(6) gene, exhibiting zero recombination in selected recombinants, was strongly associated with SW in chickpea. The genomic constitution of low- and high-SW parental accessions/NILs are represented by “A” and “B,” respectively. The genetic (cM)/physical (bp) distances and identities of the markers mapped on the LGs/chromosomes are indicated at the left and right sides of the chromosomes, respectively. The SNPs flanking and tightly linked with a CaqSW2.4 QTL and CaABCC3(6) gene mapped on chromosome 2 are indicated with blue and red lines, respectively. The digits inside square brackets denote the numbers of recombinants between CaqSW2.4 QTL/CaABCC3(6) and SNPs. B, The differential expression profile of CaABCC3(6) (validated by high-resolution GWAS, gene-by-gene regional association mapping, and map-based cloning) in vegetative (root, shoot, and leaf) and reproductive (flower, pod, embryo, cotyledon, and mature seed) tissues and four seed development stages (DS1–DS4) of 76 kb CaqSW2.4 QTL-introgressed NILs as well as mapping parental accessions (ICC 6013, ICC 14649, ICC 13764, and ICC 19192) of desi and kabuli with low and high SW. The green, black, and red in the color scale at the top signify low, medium, and high average log signal expression of genes in different tissues/stages, respectively. The accessions/NILs and tissues selected for expression profiling are indicated on the right and upper part of the expression map, respectively. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW; and NIL, near-isogenic line. DS1, 0–10 DAP; DS2, 11–20 DAP; DS3, 21–30 DAP; and DS4, 31–40 DAP.

Figure 7.

Haplotype-specific LD and association mapping in a strongly SW-associated gene, CaABCC3(6), delineated by GWAS, gene-by-gene regional association analysis and map-based cloning. Genomic organization/constitution of the CaABCC3(6) gene (A) including its (B) CDS, exhibiting the distribution of SNPs in different sequence components of this gene. C, The genotyping of 20 SNPs (A and B) in different coding and noncoding sequence components of CaABCC3(6) in all 291 cultivated (desi and kabuli) and 81 wild chickpea accessions constituted three haplotypes (D). Three haplotypes, HAP A, HAP B, and HAP C, exhibited strong association with low, medium, and high SW, respectively. The nonsynonymous and regulatory SNPs exhibiting differentiation, especially between LSWH (HAP A) and HSWH (HAP C), are highlighted in red and violet, respectively. The value r² indicates the frequency correlation between pairs of alleles across a pair of SNP loci. Boxplots for 100-SW based on three haplotypes, HAP A, HAP B, and HAP C, constituted in (E) desi (189 accessions), (F) kabuli (102), and (G) wild (81) chickpea, demonstrating their strong associations with low, medium, and high SW, respectively. Box edges represent the upper and lower quantiles, with the median value in the middle of the box. The digits within the square brackets denote the number of accessions representing each class of haplotype associated with SW. *P < 0.0001, two-sided Wilcoxon test. HAP, haplotype; LSWH/MSWH/HSWH, low-/medium-/high-SW haplotype.

Figure 8.

The differential expression profile of low- (HAP A) and high- (HAP C) SW CaABCC3(6) haplotypes of desi and kabuli in vegetative (root, shoot, and leaf) and reproductive tissues (flower, pod, embryo, cotyledon, and mature seed) and four seed development stages (DS1–DS4) of corresponding haplotype-introgressed NILs of desi and kabuli with low and high SW (A). The green, black, and red in the color scale at the top signify low, medium, and high average log signal expression values of the gene in different tissues/stages, respectively. The accessions/NILs and tissues selected for expression profiling are indicated on the right and upper part of the expression map, respectively. B, Autoradiogram depicting the northern hybridization pattern of strongly SW-associated low- (HAP A) and high- (HAP C) SW CaABCC3(6) haplotypes in seed and cotyledon of corresponding haplotype-introgressed NILs with contrasting SW. The transcript sizes (in kb) hybridizing with the probes. (I) CaABCC3[6] gene haplotypes and (II) elongation factor 1-alpha as an internal control are indicated by arrows. (III) Normalized RNA isolated from the tissues of NILs. C, Transient expression assay to determine the effects of low- (HAP A), medium- (HAP B), and high- (HAP C) SW haplotypes of desi and kabuli constituted by the regulatory SNPs from the URR of CaABCC3(6) on its expression. Left, the construct backbone with URR haplotypes (HAP A, HAP B, and HAP C) of desi and kabuli influencing the expression of the cauliflower mosaic virus (CaMV) 35S promoter-driven GUS (β-glucuronidase) reporter gene (cloned in pCAMBIA1301) along with a control CaMV 35S promoter regulating the expression of the GFP (GFP) reporter gene (cloned in pCAMBIA1302). Right, corresponding expression levels of GUS-URR haplotypes relative to control GFP in corresponding haplotype-introgressed low- and high-SW NILs of desi and kabuli. To estimate the effect of the medium-SW haplotype (HAP B) on CaABCC3(6) gene expression, four desi (ICC 8318 and ICC 12426) and kabuli accessions (ICC 7272 and ICC 7654) with medium SW were selected from a constituted association panel for a transient expression assay. Horizontal error bars signify the mean se for each construct with 18 independent replicates. Asterisk (*), Significant difference statistically at a P ≤ 0.0001 estimated by Duncan’s test. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW; LSWH/HSWH, low-/high-SW haplotype; and NIL, near-isogenic line.

Figure 9.

Schematic depiction of the strategies undertaken to develop high- and low-SW NILs of desi and kabuli chickpea by introgressing the 76-kb CaqSW2.4 QTL and target trait-specific CaABCC3(6) haplotypes from corresponding high- and low-SW parental accessions of two intraspecific mapping populations of desi (ICC 6013 × ICC 14649) and kabuli (ICC 13764 × ICC 19192) with their counterparts through marker/haplotype-assisted foreground and background selection as depicted in Supplemental Figure S8. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW; NIL, near-isogenic line; DLSWH/DMSWH/DHSWH, Desi low-/medium-/high-SW haplotype, respectively; KLSWH/KMSWH/KHSWH, Kabuli low-/medium-/high-SW haplotype; E, embryo; C, cotyledon. Scale bar = 1 cm.

Figure 10.

Variation in SW mediated through modulation of GHS content and GHS conjugates transportation during seed development stages of CaABCC3(6) gene haplotype-introgressed NILs of desi and kabuli chickpea. A, Physical estimation of variation in average weight (mg) of single seed during development stages (DS1–DS4) of seeds, including embryo and cotyledons of low- and high-SW desi and kabuli NILs as well as desi and kabuli mapping parental accessions (ICC 6013, ICC 14649, ICC 13764, and ICC 19192). Error bars represent se (n = 60). *P < 0.001, Student’s two-tailed t test. B, Variation in GSH content (nmol) measured in one gram fresh weight (FW) of seeds during the development stages (DS1–DS4) of seeds, including embryo and cotyledons of low and high SW desi and kabuli NILs as well as desi and kabuli mapping parental accessions (ICC 6013, ICC 14649, ICC 13764, and ICC 19192). Error bars represent se (n = 60). *P < 0.001, Student’s two-tailed t test. C, Complementation assay of yeast ∆ycf1 mutants by CaABCC3(6) gene haplotypes. Yeast parent strain YPH299 and its ∆ycf1 mutant was used for the complementation of high (HAP C, CaABCC3[6]^HSWH) and low (HAP A, CaABCC3[6]^LSWH) SW haplotypes constituted from a CaABCC3(6) gene. Empty vector pYES260 transformed to both the strains were used as control. After induction, an aliquot of cells at an optical density of 0.1 and subsequent 10-fold dilutions of the same were spotted on YPD plates containing (I) no CdSO₄ as control and (II) 100 µM of CdSO₄ and incubated at 30°C. Images of all the plates were captured 3 d post incubation. D, CaABCC3(6) gene haplotype-dependent uptake of ³H-GSSG and ³H-DNP-GS. The rates of high (HAP C, CaABCC3[6]^HSWH) and low SW CaABCC3[6] gene haplotype (HAP A, CaABCC3[6]^LSWH)-dependent uptake were estimated by subtracting the radioactivity acquired by vacuolar membrane-enriched vesicles prepared from pYES3-transformed cells from that acquired by the equivalent membrane fraction from pYES3-(HAP C, CaABCC3[6]^HSWH) or pYES3-(HAP A, CaABCC3[6]^LSWH)-transformed DTY168 cells. Error bars represent se (n = 3). *P < 0.001, Student’s two-tailed t test. DS1, 0–10 DAP; DS2, 11–20 DAP; DS3, 21–30 DAP; and DS4, 31–40 DAP. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW; DLSWH/DMSWH/DHSWH, Desi low-/medium-/high-SW haplotype; KLSWH/KMSWH/KHSWH, Kabuli low-/medium-/high-SW haplotype.

Figure 11.

Modulation of GHS conjugates transportation by the superior haplotype of an ABCC3 transporter, CaABCC3(6), enhances SW/yield and seed quality in the corresponding gene haplotype-introgressed NILs of desi and kabuli as compared to high-yielding Indian chickpea cultivars. A, Picture illustrating the comparative overview of seed size/SW variation observed between low- and high-SW NILs as well as high-yielding Indian varieties of desi and kabuli chickpea. The details of agronomic characteristics of these NILs and Indian varieties are mentioned in Supplemental Table S18. B, Diagrammatic representation of a plant cell playing the role of the ABCC transporter. The high SW (100-SW haplotype [HAP C, ABCC3(6)^HSWH] exhibited a greater fold of transcript expression in the seed cotyledons compared to its low SW counterpart [HAP A, ABCC3(6)^LSWH]). At the cellular level, the ABCC3 transporter is localized in the vacuolar membrane where it is involved in the transportation of GSSG, and glutathione-conjugates (GS-X) from the cytosol to the vacuole. GSSG and other GS-Xs are derived from cytosolic redox conversion of GHS by several enzymes like glutathione reductase (GR), glutathione-s-transferase tau 24 (GST), and glutathione peroxidase 4 (GPX4). These GHS conjugates once inside the vacuole are degraded to their component amino acids by gamma-glutamyl transpeptidase (GGT), which may then be utilized by the cell for its growth and development. The GSSG and GS-X are transported out of the cell by oligopeptide transporters (OPTs), and the same process of breakdown of these conjugates by GGT also takes place in the apoplast. The high-SW haplotype (HAP C, ABCC3[6]^HSWH) as compared to low SW haplotype (HAP A, ABCC3[6]^LSWH) is more efficient in the vacuolar transport of GSSG and GHS-conjugate like DNP-GS as well as their subsequent metabolism in the cell through transcriptional regulation by inducing transcript expression and accumulation in the vacuoles of seeds and cotyledons at the mature stage. This overall enhances the growth, development, and maturation of seeds and cotyledons in high- vis-à-vis low-SW haplotype-introgressed NILs and thereby increases the thiolic amino acid (Met and Cys) and protein contents in seeds of desi and kabuli chickpea. Scale bar = 1 cm. DLSW/KLSW, Desi/kabuli low SW; DHSW/KHSW, Desi/kabuli high SW; LSWH/HSWH, low-/high-SW haplotype.

Figure 12.

Summary of the delineation of an ABCC transporter gene regulating SW in chickpea and subsequent introgression of the superior haplotype of this gene to improve a desi variety. A, Utilization of multienvironment field phenotyping and high-resolution marker (SNP) genotyping data in an integrated molecular genetics and genomics strategy identified CaABCC3(6) and its constituted two gene haplotypes as key regulators of SW in chickpea. B, Marker (haplotype)-assisted introgression of superior high SW CaABCC3(6) gene haplotype (allele) into a desi variety (ICCV 93954) leads to overall increase in SW/yield, productivity, and protein content.