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.


CONCLUSIONS
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.
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, Canada. ksferrar@ucalgary.ca.
Kiva
K
Ferraro
Plant BioSystems, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. ljin1@ualberta.ca.
Alena L
AL
Jin
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, Canada. nguyendt@ucalgary.ca.
Trinh-Don
TD
Nguyen
Plant BioSystems, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. dennis.reinecke@ualberta.ca.
Dennis M
DM
Reinecke
Plant BioSystems, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. jocelyn.ozga@ualberta.ca.
Jocelyn A
JA
Ozga
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, Canada. daekyun.ro@ucalgary.ca.
Dae-Kyun
DK
Ro
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D044945:Proanthocyanidins / Q000096:biosynthesis*; D012639:Seeds / Q000201:enzymology*; D059467:Transcriptome
D010940:Plant Proteins; D044945:Proanthocyanidins; C013221:proanthocyanidin; D010088:Oxidoreductases