Genomic Analysis of Storage Protein Deficiency in Genetically Related Lines of Common Bean (Phaseolus vulgaris)

Abstract

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

Keywords: Phaseolus vulgaris; common bean; deletion; genome sequencing; introgression; lectin; phaseolin.

FIGURE 1

(A) Affinity purification of mannose lectin FRIL. SDS-PAGE of mature seed extracts after purification on mannose agarose. The band of 20 kDa corresponds to the N-terminal subunit of mannose lectin FRIL. The bands of 17 and 16 kDa correspond to the C-terminal subunit. The identity of protein bands was confirmed by proteomics (see Table 2). (B) Immunoblotting of total protein extracts from mature seed with polyclonal antibodies raised against recombinant α-amylase inhibitor. Bands of 14.5 and 13 kDa correspond to chain 2 and chain 1 of α-amylase inhibitor 1, respectively.

FIGURE 2

(A) Analysis of lectin gene composition by genomic PCR. The presence or absence of lectin genes was evaluated using gene-specific primers. The phaseolin-deficient P. coccineus accession is distinct from the one used by Osborn et al. (2003). (B) Sequence alignment of conceptual translations of PCR products coding for mannose lectin FRIL.

FIGURE 3

(A) Sequence reads of lectin locus aligned to the reference BAT-93 genome. Paired end reads from the four genotypes sequenced by Illumina HiSeq2000/2500 were aligned and visualized with IGV. The positions of the lectin genes are indicated by boxes. Gray indicates sequence identity. Color highlights variant bases compared to the reference sequence; Green for A; Red for T; Orange for G; and Blue for C. PDLEC2 and α-amylase inhibitor like protein gene (α-AI-like) are not covered in Sanilac; likewise α-amylase inhibitor 1 (α-AI1) in SARC1 and SMARC1-PN1. BAT-93 and SMARC1N-PN1 have the pdlec1 allele in place of pha-L. (B) Schematic view of polymorphisms in the promoter of α-amylase inhibitor like protein between SMARC1N-PN1 versus SARC1 and SMARC1-PN1 giving rise to unique cis-regulatory elements in SMARC1N-PN1. Analysis of cis-regulatory elements was performed using the Place database. C, CAAACAC element; S, soybean embryo factor 4 binding motif; A, core AACA motif; ABRE, abscisic acid related element.

FIGURE 4

(A) Schematic view of cis-regulatory elements present in the proximal promoter of β-phaseolin in SARC1 and Sanilac versus SMARC1N-PN1 and SMARC1-PN1. Regulatory motifs are designated as in Chandrasekharan et al. (2003). A single nucleotide polymorphism converts a G-box in SARC1 and Sanilac to an ACGT motif in SMARC1N-PN1 and SMARC1-PN1 (B). G, G-box; E, E-box; C, CACA box; V, vicillin box; CCA, CCAAT box. (C) Sequence read alignments to the α-phaseolin gene from Sanilac (accession number X52626). The bar below shows the intron-exon structure with exons numbered as black boxes. The alignment shows incomplete coverage of the promoter region in SMARC1-PN1 and SMARC1N-PN1.