Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis
Postharvest seed coat darkening in pinto bean is an undesirable trait resulting in a loss in the economic value of the crop. The extent of darkening varies between the bean cultivars and their storage conditions.
Metabolite analysis revealed that the majority of flavonoids including proanthocyanidin monomer catechin accumulated at higher level in a regular darkening (RD) pinto line CDC Pintium than in a slow darkening (SD) line 1533–15. A transcriptome analysis was conducted to compare gene expression between CDC Pintium and 1533–15 and identify the gene (s) that may play a role in slow darkening processes in 1533–15 pinto.
RNAseq against total RNA from RD and SD cultivars found several phenylpropanoid genes, metabolite transporter genes and genes involved in gene regulation or modification to be differentially expressed between CDC Pintium and 1533–15.
RNAseq analysis and metabolite data of seed coat tissue from CDC Pintium and 1533–15 revealed that the whole proanthocyanidin biosynthetic pathway was downregulated in 1533–15. Additionally, genes that encode for putative transporter proteins were also downregulated in 1533–15 suggesting both synthesis and accumulation of proanthocyanidin is reduced in SD pintos.
KeywordsPinto bean Postharvest seed coat darkening Proanthocyanidins Transcriptome Phenylpropanoids
Postharvest darkening of seed coat is a concern for dry bean (Phaseolus vulgaris L.) producers worldwide. The seed coat of many beans such as pinto, cranberry and red beans darken during aging which affects their visual quality, leading to a decrease in consumer preference due to which the common bean producers, exporters and vendors encounter significant loss in crop value. Studies have indicated that the postharvest darkening phenomenon is attributed to a combination of environmental factors such as elevated temperatures, humidity, exposure to light [1, 2] as well as crop genetics [3, 4]. There are at least 3 phenotypes of bean seed coats that respond differently to aging as identified by various common bean breeding programs: regular darkening (RD), slow darkening (SD) and non-darkening (ND) .
An attempt to study the genetics of postharvest seed coat darkening has suggested that this trait is controlled by at least two unlinked genes in dry beans. The J locus determines the tendency to darken such that a homozygous recessive (jj) results in an ND phenotype. The gene J is epistatic to a second major gene Sd, that determines how rapidly a seed coat will darken . A simple sequence repeat (SSR) assay in a RIL mapping population generated from CDC Pintium and 1533–15 placed the Sd gene between the SSR markers Pvsd-1157 and Pvsd-1158 on chromosome 7 . However, the possibility of this trait being regulated by a number of other genes cannot be overlooked. These genes could either be present on the same chromosome as the SSR markers or on different chromosomes. A detailed study of the seed coat transcriptome across seed developmental stages could shed light on the genetic factors affecting seed coat darkening.
Here we apply a transcriptomic analysis to identify genes that are expressed differentially in CDC Pintium and 1533–15 during seed coat development and investigate their potential role in the active accumulation of proanthocyanidins and/or their regulators. Genes that might have roles in the translocation of proanthocyanidin monomers/oligomers from cytoplasm to vacuoles or vacuoles to apoplast have also been identified, which potentially provides further insight into seed coat darkening and assists in the selection of candidate genes. Our study identifies several phenylpropanoid and transporter genes that are differentially expressed (DE) between RD and SD cultivars and may be key genes in determining postharvest seed coat darkening in pinto beans.
Pinto bean (Phaseolus vulgaris L.) cultivars CDC Pintium and 1533–15 were used for the study. CDC Pintium is a rapid darkening, early maturing pinto bean cultivar developed by the University of Saskatchewan. It has a good seed coat color at harvest which turns from creamy white to dark brown in storage. 1533–15 is a slow darkening, early maturing F4−derived line developed from a cross between CDC Pintium and a breeding line SC11743–3 from the International Centre for Tropical Agriculture, and registered as CDC WM-1 . Its seed coat turns from creamy white to light brown at a much slower rate than CDC Pintium. Both cultivars take 20-21 days for flowering.
To collect seed coat samples, CDC Pintium and 1533–15 were grown in Pro-Mix PGX in a growth chamber under a 16 h light at 25 °C and 8 h dark at 20 °C cycle with 70–80% relative humidity. Light intensity was maintained at 300–400 μmol photons/m2/s. Developing seeds were harvested at four different developmental stages (30, 50, 150, 350 mg seed weight) at the same time of the day (2 pm), and their seed coats collected. The seed coat tissues were immediately frozen in liquid nitrogen and stored at − 80 °C.
Total RNA was extracted from pinto bean seed coat tissues (30, 50, 150, 350 mg seed weight) using a modified LiCl method . RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA), and their integrity checked using a 2100 Bioanalyzer (Agilent Technologies, USA).
RNAseq and data analysis
The seed coat mRNA from CDC Pintium and 1533–15 (150 mg stage) was sequenced using a HiSeq2000 (Illumina Inc., USA) at the National Research Council (Saskatoon, Canada). Four biological replicates per cultivar were sequenced using 100 bp paired-end runs. Additional adapter trimming (additional to that performed by the sequencer) was performed using a custom Perl script against adapter sequences identified using FastQC. Reads from each biological replicate were mapped to representative transcripts of the P. vulgaris genome (Phytozome release V2.1, https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvulgaris, “PrimaryTranscripOnly” file)  using BWA with 3′ end trimming (base quality > 30) . SAMtools was then used to filter PCR duplicates, remove reads with low mapping quality (Q < 20) and extract the total number of uniquely mapped reads per transcript . Read counts from SAMtools were imported into R and normalized using the DESeq “counts” function. Low expressing genes possessing one or zero read counts when summed across all treatments were filtered by removing the bottom 10% quantile of genes using the R quantile function. These low expressors introduce division by zero errors in downstream analysis and are therefore dropped. Differential expression was assessed using the negative binomial test (FDR < 0.001) of DESeq . Heatmaps were generated in R using the heatmap.2 function.
Gene ontology enrichment and analysis
DE genes were subjected to Gene Ontology (GO) enrichment analysis using the Singular Enrichment Analysis tool available on AgriGO v2.0 (http://bioinfo.cau.edu.cn/agriGO/analysis.php) with a significance level of 5% using Fisher statistical testing and Yekutieli multi-test adjustment.
Total RNA from each sample was treated with DNaseI to remove contaminating DNA using the TURBO DNA-free™ kit (Life Technologies, USA). Total RNA (1 μg) from each sample was used for cDNA synthesis using the Thermoscript™ RT-PCR System (Invitrogen, USA). For quantitative RT-PCR, SsoFastTM EvaGreen® Supermix (Bio-Rad, USA) was used with the CFX96 real-time PCR detection system (Bio-Rad, USA). The primer combination and amplicon sizes are shown in Additional file 1: Table S1. The amplicon was cloned into pGEM-T Easy vector (Promega, USA), and its sequence verified. P. vulgaris ubiquitin (Phvul.007G052600) was used as a reference gene for data normalization and to calculate the relative mRNA levels. Two biological replicates per cultivar and three technical replicates per biological replicates were used for qPCR analysis using CFX Manager (Bio-Rad, USA).
Extraction and analysis of flavonoids
Extraction of polyphenols from pinto bean seed coat was carried out according to Hu et al.  with some modifications at The Metabolomics Innovation Centre, University of Alberta. The ground seed coat powder (1 g) samples were extracted in 10 mL of methanol: water (80:20 v/v) containing 1% HCl. The samples were first sonicated at ambient temperature for 30 min, then at 40 °C for another 30 min followed by incubation in boiling water bath for 30 min with regular vortexing. The extracts were cooled, shaken at 300 rpm for 4 h and centrifuged at 3000 rpm for 20 min at ambient temperature. The supernatant was filtered under vacuum at room temperature and the filtrate lyophilized after purging under nitrogen gas for 30 min. The lyophilized powder was dissolved in 25% methanol.
HPLC analysis of the methanol extracts was performed using an Agilent G1311 A quarternary 1100 series HPLC pump (Agilent Technologies), a Synergi RP-polar (250 × 4.6 mm, 4 μm) C18 column (Phenomenex) connected to an Agilent G1315B diode array detector. Data were collected and analyzed using ChemStation software (Agilent Technologies). Phenolic compounds in the extracts were analyzed using the reference HPLC method  with gradient elution program [solution A, 50 mM sodium phosphate pH 2.5 (by the addition of 85% ortho phosphoric acid), solution B, 100% methanol]: 0 min, 5% B; 15 min, 30% B; 40 min, 40% B; 60 min, 50% B; 65 min, 55% B; 80 min, 100% B; 85 min, 5% B and 90 min, 5% B. The flow rate was 1.0 mL/min and injection volume was 40 μL. Absorbance was detected at 254, 280, 306 and 340 nm using the Agilent diode array detector. Phenolic compounds in the extracts were identified by comparison of their retention times with the spectra of known polyphenol standards and quantified by using the calibration curve for each phenolic compound obtained by peak areas from the chromatogram.
Results and discussion
Flavonoid analysis of immature seed coat of CDC Pintium and 1533–15
Analysis of seed coat transcriptome in SD and RD pinto beans
RNAseq quality and coverage of bean transcriptome
Q30 bases (%)
Uniquely mapped reads
Genes hit (%)
Differential gene expression analysis
Before calculating differential expression, an independent filter removing the bottom 10% of transcripts based on total read counts (3186 transcripts including the 2432 genes with no reads mapped mentioned above) was applied in order to simplify the analysis. At a false discovery rate (FDR) of ≤0.001, a total of 922 genes were DE between the two pinto bean lines with 260 genes up-regulated (at least 2× higher) and 203 genes down-regulated (less than 2× lower) in CDC Pintium compared to 1533–15 seed coats (Additional file 3: Table S2). On examination of the Benjamini-Hochberg adjusted p-value distribution (Additional file 4: Figure S2A), and the log p-value as a function of all genes ranked by total read counts (Additional file 4: Figure S2B), a large percentage of highly significant genes and a very strong baseline of moderately significant genes were observed. Grouping of log significance scores into 3 levels of significance categorized the DE genes with a strong baseline of 814 significant transcripts (p ≤ 0.001), 95 highly significant transcripts (p ≤ 1e-15) and a collection of 13 extremely significant transcripts (p ≤ 1e-55) (Additional file 5: Figure S3, Additional file 6: Table S3). As the two lines studied, CDC Pintium and 1533–15, are closely related but not genetically identical, it is possible that the 814 significant transcripts in the lowest level represent expression variation that exists across the baseline functions of seed coat biology between the two varieties that may include the genes that are involved in the slow darkening trait. Further, as the primary phenotypic difference between the two genotypes is the rate at which seed coat darkening occurs, the hypothesis is made that the upper two levels of significant transcripts constitute, at least in part, the components of the metabolic pathways necessary for producing this difference. Regardless, it is important to note that the gene expression observed here in the young seed coat leads to a flavonoid profile that produces the trait postharvest.
List of extremely significantly (p ≤ 1e-55) differentially expressed genes between CDC Pintium and 1533–15 at stage 150 mg of seed development
Expression analysis of phenylpropanoid genes
The dataset generated in this study provides a significant resource for further molecular and biochemical studies of postharvest seed coat darkening in pinto beans.
The authors thank Ling Chen, Aga Pajak, Nishat Shayala Islam and Alex Molnar for technical assistance.
This research was supported by Agriculture and Agri-Food Canada’s Abase grant to SD. Funding authority was not involved in both experimental design and execution of this study.
Availability of data and materials
All data generated during this study are included in this published article and its supplementary additional files. RNAseq data are available in the European Nucleotide Archive with the accession number PRJEB22986.
KD and RSA conducted the experiments, analyzed the data and wrote the draft manuscript, HRM conducted qPCR experiments, analyzed the data and involved in draft manuscript preparation, KB provided the germplasms and contributed to manuscript preparation, FM contributed to experimental design and manuscript preparation, and SD conceived and designed experiments, supervised all aspects of the project and prepared final draft manuscript. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Pinto bean seeds used in this study was supplied by Dr. K. Bett, University of Saskatchewan. Plants were grown in the growth room for sample collection. The research conducted in this study required neither approval from an ethics committee, nor involved any human or animal subjects.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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