QUES, a new Phaseolus vulgaris genotype resistant to common bean weevils, contains the Arcelin-8 allele coding for new lectin-related variants

In common bean (Phaseolus vulgaris L.), the most abundant seed proteins are the storage protein phaseolin and the family of closely related APA proteins (arcelin, phytohemagglutinin and α-amylase inhibitor). High variation in APA protein composition has been described and the presence of arcelin (Arc) has been associated with bean resistance against two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven Arc variants have been identified, all in wild accessions, however, only those containing Arc-4 were reported to be resistant to both species. Although many efforts have been made, a successful breeding of this genetic trait into cultivated genotypes has not yet been achieved. Here, we describe a newly collected wild accession (named QUES) and demonstrate its resistance to both A. obtectus and Z. subfasciatus. Immunological and proteomic analyses of QUES seed protein composition indicated the presence of new Arc and arcelin-like (ARL) polypeptides of about 30 and 27 kDa, respectively. Sequencing of cDNAs coding for QUES APA proteins confirmed that this accession contains new APA variants, here referred to as Arc-8 and ARL-8. Moreover, bioinformatic analysis showed the two proteins are closely related to APA components present in the G12949 wild bean accession, which contains the Arc-4 variant. The presence of these new APA components, combined with the observations that they are poorly digested and remain very abundant in A. obtectus feces, so-called frass, suggest that the QUES APA locus is involved in the bruchid resistance. Moreover, molecular analysis indicated a lower complexity of the locus compared to that of G12949, suggesting that QUES should be considered a valuable source of resistance for further breeding purposes

Antinutritional factors in pearl millet grains: Phytate and goitrogens content variability and molecular characterization of genes involved in their pathways.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important "orphan" cereal and the most widely grown of all the millet species worldwide. It is also the sixth most important cereal in the world after wheat, rice, maize, barley, and sorghum, being largely grown and used in West Africa as well as in India and Pakistan. The present study was carried out in the frame of a program designed to increase benefits and reduce potential health problems deriving from the consumption of pearl millet. The specific goal was to provide a database of information on the variability existing in pearl millet germplasm as to the amounts of phytate, the most relevant antinutrient compound, and the goitrogenic compounds C-glycosylflavones (C-GFs) accumulated in the grain.Results we obtained clearly show that, as indicated by the range in values, a substantial variability subsists across the investigated pearl millet inbred lines as regards the grain level of phytic acid phosphate, while the amount of C-GFs shows a very high variation. Suitable potential parents to be used in breeding programs can be therefore chosen from the surveyed material in order to create new germplasm with increased nutritional quality and food safety. Moreover, we report novel molecular data showing which genes are more relevant for phytic acid biosynthesis in the seeds as well as a preliminary analysis of a pearl millet orthologous gene for C-GFs biosynthesis. These results open the way to dissect the genetic determinants controlling key seed nutritional phenotypes and to the characterization of their impact on grain nutritional value in pearl millet

Calcium redistribution contributes to the hard-to-cook phenotype and increases PHA-L lectin thermal stability in common bean low phytic acid 1 mutant seeds

Seed phytic acid reduces mineral bioavailability by chelating minerals. Consumption of common bean seeds with the low phytic acid 1 (lpa1) mutation improved iron status in human trials but caused adverse gastrointestinal effects, presumably due to increased stability of lectin phytohemagglutinin L (PHA-L) compared to the wild type (wt). A hard-to-cook (HTC) defect observed in lpa1 seeds intensified this problem. We quantified the HTC phenotype of lpa1 common beans with three genetic backgrounds. The HTC phenotype in the lpa1 black bean line correlated with the redistribution of calcium particularly in the cell walls, providing support for the "phytase-phytate-pectin" theory of the HTC mechanism. Furthermore, the excess of free cations in the lpa1 mutation in combination with different PHA alleles affected the stability of PHA-L lectin

Percentage contribution of each C-GF to the total C-GFs contained in the grains of 96 pearl millet lines from a panel of inbred lines covering a large genetic diversity.

<p>Vitexin, yellow; glucosyl vitexin, orange; rhamnosyl vitexin, dark red; orientin, light green; glucosyl orientin, green; rhamnosyl orientin, blue.</p

Distribution of phytic acid content, total phosphorus and inorganic phosphate in the grains of the first and second generations of inbred lines contrasting for phytic acid and total C-GFs content.

<p>ICML157032, lpa and hcgf and ICML157101, hpa and lcgf. Each data is the mean of two biological replicates.</p

Distribution of C-GFs content among the grains of 96 pearl millet lines from a panel of inbred lines covering a large genetic diversity.

<p>Red arrowheads indicate the most contrasting lines, ICML157013 and ICML15711, having the highest and lowest C-GFs content, respectively. Red boxes and arrows indicate the two inbred lines selected for their contrasting content of phytic acid. Each bar is the sum of the content of each C-GF (vitexin, yellow; glucosyl vitexin, orange; rhamnosyl vitexin, dark red; orientin, light green; glucosyl orientin, green; rhamnosyl orientin, blue) in each line.</p

Expression analysis by quantitative RT-PCR of genes involved in phytic acid pathway in leaves, stems, plantlets and anthesis flowers of the reference line SDEB4L-160P6.

<p>Color of the bars indicate different organs: dark green, leaf; yellow, stem; light green, plantlet; orange, flower. (A) expression of <i>PglMIPS_337</i> and <i>PglMIPS_896</i>; (B) expression of <i>PglMIK</i>; (C) expression of <i>PglIMP;</i> (D) expression of <i>PglITPK_980</i>, <i>PglITPK_602</i>, <i>PglITPK_425</i> and PglITPK_775; (E) expression of <i>PglIPK1</i>; (F) expression of <i>PglMRP</i>; (G) expression of <i>PglIPK2</i>. Leaf samples were used as calibrators.</p

Distribution of phytate and total phosphate among 145 pearl millet lines from a panel of inbred lines covering a large genetic diversity.

<p>Phytic acid phosphorus (PAP, blue line), total phosphorus (P_tot, red line), ratio of phytic acid content to total phosphorus (PAP/P_tot,, violet line) and P_i (green line). Red dots and boxes indicate the most contrasting lines for phytic acid content.</p

Neighbour-joining trees inferred from the deduced amino acid sequence of intermediate-late phytic acid pathway genes identified in pearl millet.

<p><b>(A) ITPK, (B) IPK2, IPK1 (C)</b>. Bars indicate the number of amino acid substitutions per site. Sequences IDs are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198394#pone.0198394.s002" target="_blank">S2 Table</a>.</p