Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds

BACKGROUND: Proanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied. RESULTS: PAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. ‘Courier’ and ‘Solido’ PAs were primarily prodelphinidin-types, whereas the PAs from ‘LAN3017’ were mainly the procyanidin-type. The mean degree of polymerization of ‘LAN3017’ PAs was also higher than those from ‘Courier’ and ‘Solido’. Next-generation sequencing of ‘Courier’ seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis. CONCLUSION: Significant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-014-0238-y) contains supplementary material, which is available to authorized users

Neglecting legumes has compromised human health and sustainable food production

The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner 'nutritious seeds for a sustainable future'. A second green revolution is required to ensure food and nutritional security in the face of global climate change. Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming. In addition, a legume-rich diet has health benefits for humans and livestock alike. However, grain legumes form only a minor part of most current human diets, and legume crops are greatly under-used. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. The current lack of coordinated focus on grain legumes has compromised human health, nutritional security and sustainable food production.We thank World University Network (WUN) and research collaboration awards (UWA and the University of Leeds) for financial support. CHF thanks the Biotechnology and Biological Sciences Research Council (BBSRC UK (BB/M009130/1) and the European Union (KBBE-2012-6-311840; ECOSEED) for financial support. JWC thanks BBSRC (UK) and Wherry and Sons, (UK) for an industrial CASE studentship (BB/K501839/1). H-ML was supported by the Hong Kong RGC Collaborative Research Fund (CUHK3/CRF/11G), the Lo Kwee-Seong Biomedical Research Fund and Lee Hysan Foundation. Ka-Ming Fung, Qianwen Wang, Lydia Kit-Wah Siu, and Yihan Jiang of The Chinese University of Hong Kong assisted in the production of Fig. 2, Table 1 and the associated webpage, highlight and cover design. We thank Hari Upadhyaya for the images shown in Fig. 4. TAM and JMH thank the Western Australian Government, Department of Industry and Resources for financial support. The authors thank Bodhi‟s Bakery, Fremantle, Western Australia, for baking the bread and biscuits and Belmar Foods, Balcatta, Western Australia, for manufacturing and providing the pasta. BNK and HB are supported by Australian Research Council (ARC), ITRH – Legumes for Sustainable Agriculture (IH140100013). MJC and CHF thank the ARC (DP150103211) for financial support. AJM is supported by grant funding (BB/JJ004553/1 and BB/L010305/1) from the BBSRC and the John Innes Foundation.http://www.nature.com/nplantshb2017Plant Production and Soil Scienc

Pollination-, Development-, and Auxin-Specific Regulation of Gibberellin 3β-Hydroxylase Gene Expression in Pea Fruit and Seeds

To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (−2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6–10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth

Developmental and Embryo Axis Regulation of Gibberellin Biosynthesis during Germination and Young Seedling Growth of Pea

The expression patterns of five genes (PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2) encoding five regulatory gibberellin (GA) biosynthesis enzymes (two GA 20-oxidases, a GA 3β-hydroxylase, and two GA 2β-hydroxylases) were examined to gain insight into how these genes coordinate GA biosynthesis during germination and early postgermination stages of the large-seeded dicotyledonous plant pea (Pisum sativum). At the time the developing embryo fills the seed coat, high mRNA levels of PsGA20ox2 (primarily responsible for conversion of C20-GAs to GA(20)), PsGA2ox1 (primarily responsible for conversion of GA(20) to GA(29)), and PsGA2ox2 (primarily responsible for conversion of GA(1) to GA(8)) were detected in the seeds, along with high GA(20) and GA(29) levels, the enzymatic products of these genes. Embryo maturation was accompanied by a large reduction in PsGA20ox2 and PsGA2ox1 mRNA and lower GA(20) and GA(29) levels. However, PsGA2ox2 transcripts remained high. Following seed imbibition, GA(20) levels in the cotyledons decreased, while PsGA3ox1 mRNA and GA(1) levels increased, implying that GA(20) was being used for de novo synthesis of GA(1). The presence of the embryo axis was required for stimulation of cotyledonary GA(1) synthesis at the mRNA and enzyme activity levels. As the embryo axis doubled in size, PsGA20ox1 and PsGA3ox1 transcripts increased, both GA(1) and GA(8) were detectable, PsGA2ox2 transcripts decreased, and PsGA2ox1 transcripts remained low. Cotyledonary-, root-, and shoot-specific expression of these GA biosynthesis genes and the resultant endogenous GA profiles support a key role for de novo GA biosynthesis in each organ during germination and early seedling growth of pea

Impact of Susceptibility on Plant Hormonal Composition during Clubroot Disease Development in Canola (<i>Brassica napus</i>)

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease of crucifers associated with the formation of large root galls. This root enlargement suggests modulation of plant hormonal networks by the pathogen, stimulating cell division and elongation and influencing host defense. We studied physiological changes in two Brassica napus cultivars, including plant hormone profiles—salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), the auxin indole-3-acetic acid (IAA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)—along with their selected derivatives following inoculation with virulent and avirulent P. brassicae pathotypes. In susceptible plants, water uptake declined from the initial appearance of root galls by 21 days after inoculation, but did not have a significant effect on photosynthetic rate, stomatal conductance, or leaf chlorophyll levels. Nonetheless, a strong increase in ABA levels indicated that hormonal mechanisms were triggered to cope with water stress due to the declining water uptake. The free SA level in the roots increased strongly in resistant interactions, compared with a relatively minor increase during susceptible interactions. The ratio of conjugated SA to free SA was higher in susceptible interactions, indicating that resistant interactions are linked to the plant’s ability to maintain higher levels of bioactive free SA. In contrast, JA and its biologically active form JA-Ile declined up to 7-fold in susceptible interactions, while they were maintained during resistant interactions. The ACC level increased in the roots of inoculated plants by 21 days, irrespective of clubroot susceptibility, indicating a role of ethylene in response to pathogen interactions that is independent of disease severity. IAA levels at early and later infection stages were lower only in susceptible plants, suggesting a modulation of auxin homeostasis by the pathogen relative to the host defense system

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit1[OA]

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA20 to bioactive GA1) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA20 to GA29), suggesting a concerted regulation to increase levels of bioactive GA1 following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA1 to GA8) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA1, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [14C]GA12 to [14C]GA1 only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA1 required for initial fruit set and growth

Tissue-Specific Regulation of Gibberellin Biosynthesis in Developing Pea Seeds1[W][OA]

Previous work suggests that gibberellins (GAs) play an important role in early seed development. To more fully understand the roles of GAs throughout seed development, tissue-specific transcription profiles of GA metabolism genes and quantitative profiles of key GAs were determined in pea (Pisum sativum) seeds during the seed-filling development period (8–20 d after anthesis [DAA]). These profiles were correlated with seed photoassimilate acquisition and storage as well as morphological development. Seed coat growth (8–12 DAA) and the subsequent dramatic expansion of branched parenchyma cells were correlated with both transcript abundance of GA biosynthesis genes and the concentration of the growth effector GA, GA1. These results suggest GA1 involvement in determining the rate of seed coat growth and sink strength. The endosperm’s PsGA20ox transcript abundance and the concentration of GA20 increased markedly as the endosperm reached its maximum volume (12 DAA), thus providing ample GA20 substrate for the GA 3-oxidases present in both the embryo and seed coat. Furthermore, PsGA3ox transcript profiles and trends in GA1 levels in embryos at 10 to 16 DAA and also in embryo axes at 18 DAA suggest localized GA1-induced growth in these tissues. A shift from synthesis of GA1 to that of GA8 occurred after 18 DAA in the embryo axis, suggesting that deactivation of GA1 to GA8 is a likely mechanism to limit embryo axis growth and allow embryo maturation to proceed. We hypothesize that GA biosynthesis and catabolism are tightly regulated to bring about the unique developmental events that occur during seed growth, development, and maturation

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism1[W][OA]

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3′-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3′5′-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis

Developmental Profile of Anthocyanin, Flavonol, and Proanthocyanidin Type, Content, and Localization in Saskatoon Fruits (Amelanchier alnifolia Nutt.)

Saskatoons (Amelanchier alnifolia Nutt.) are small fruits that contain substantial quantities of flavonoids. To further characterize and understand the role of these flavonoids during fruit development, anthocyanins, flavonols, and proanthocyanidins were identified, quantified, and localized over development in cultivars that produce blue-purple or white fruit at maturity. Anthocyanin content was low in young fruit and then dramatically increased as the fruit transitioned into ripening only in the pigmented-fruit (blue-purple) cultivars. Proanthocyanidins with both A-type and B-type linkages were detected in fruit, with (−)-epicatechin as the most abundant proanthocyanidin subunit. Flavonol and proanthocyanidin content was high in, and localized throughout, the tissues of young fruit and in the developing seed coats, with levels decreasing as the fruit expanded. Our data show that flavonoid type, content, and tissue localization vary throughout development in saskatoon fruit. These data can be used to target specific fruit developmental stages and flavonoid classes for optimization of health-beneficial flavonoid content