The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication

Abstract

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy.We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas.The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity.Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.

The giant diploid faba genome unlocks variation in a global protein crop

“…4a) and content in oligomeric phenolic compounds at the hilum surface in faba bean (Fig. 4c and Extended Data Figure 13) were very similar to those observed in pea 32 . Together with the genetic data, this indicates that differential PPO activity is responsible for hilum colour variation in both pea and faba bean, but it was unclear which faba bean PPO(s) may be causative.…”

“…To examine hilum morphology, seed coat containing hilum from inbred lines Hedin/2 (dark hilum) and Tiffany (pale hilum) were dissected from mature dry seed, saturated with 2% sucrose solution under vacuum for 1 hour and embedded in cryo-gel media (Cryo-gel Leica). Samples were cut in cryotome (Leica CM1950, US) into 15µm transversal sections) and stained with Toluidine blue O (0.01 %, w/v in water; Sigma Aldrich, CZ) as previously described 46, 47 . Observation and photography was done on an Olympus BX 51 microscope (Olympus Corp., Tokyo, Japan) in bright field and figures were documented with an Apogee U4000 digital camera (Apogee Imaging Systems, Inc., Roseville, CA, USA).…”

“…Observation and photography was done on an Olympus BX 51 microscope (Olympus Corp., Tokyo, Japan) in bright field and figures were documented with an Apogee U4000 digital camera (Apogee Imaging Systems, Inc., Roseville, CA, USA). For the investigation of metabolite content of surface layers of the hilum by laser desorption-ionization imaging mass spectrometry (LDI-MS), seeds were mechanically cracked and hila with small part of surrounding tissue were separated from the rest of seed coats using microscissors (MicroSupport, Shizuoka, Japan), fixed using a double-sided tape on MALDI plates with outer surfaces facing up and analysed as previously described 46, 48 . LDI-MSI experiments were done using a high-resolution tandem mass spectrometer (HRTMS) Synapt G2-S (Waters, Milford, USA).…”

“…Samples were cut in cryotome (Leica CM1950, US) into 15µm transversal sections) and stained with Toluidine blue O (0.01 %, w/v in water; Sigma Aldrich, CZ) as previously described 46,47 .…”

“…We found the most highly associated GWA marker in a polyphenol oxidase (PPO) gene residing in a cluster of eight fully intact and highly conserved PPO genes in the ‘Hedin/2’ assembly. In pea, PPO variation controls hilum colour, and a frameshifted, non-functional form of the single PPO copy residing at the syntenic Pl locus conferring a pale hilum is fixed in all modern pea varieties 32 . The pattern of pigmentation (Fig.…”