Canadian Journal of Plant Science 591-600 (2017)
The molecular basis for an ancient colour mutant in sweet pea (Lathyrus odoratus)
Xinxin Xue, Quentin C.B. Cronk

The classic A1 locus in sweet pea (Lathyrus odoratus) was investigated by Bateson, Punnett, and Saunders in the early 20th century history of Mendelian genetics. The mutation, in the form of the pink and white cultivar 'Painted Lady', is known from the 18th century. We show that this locus is associated with a single base pair mutation (332 G/A) in the flavonoid 3',5'-hydroxylase (F3'5'H) gene. This results in an amino acid change (111 glycine/aspartic acid) in the conserved substrate recognition site 1 (SRS1) of the enzyme. The mutant flower lacks the blue pigment delphinidin and is thus pink and white, rather than purple and blue as in the wild-type. This single amino acid change at a functionally important site appears to convert the enzyme from primary F3'5'H activity to a relatively efficient F3'H, as suggested by heterologous transformation into Arabidopsis PAP1D (a mutant line that produces anthocyanin constitutively).

Fig. 1. Top panel: (A) Lathyrus odoratus wild-type cultivar 'Cupani' ('CP') and (B) pink mutant 'Painted Lady' ('PL').
Bottom panel: transverse section of the standard petal showing an epidermal location of the anthocyanins in (C) 'CP' and (D) 'PL'. 200x magnification.

Alteration of the SRS1 domain of sweet pea F3'5'H greatly increased the accumulation of cyanidin in a heterologous system
The cyanidin content in the mutant 'PL'-F3'5'H transgenic lines was not only 5–6 times that of the control, it was also considerably higher than the wild-type 'CP'-F'3'5'H transgenic lines. All F3'5'H enzymes have some F3'H activity (Seitz et al. 2006), but it is remarkable that the alteration of a single amino acid in the F3'5'H apparently increases the propensity of the enzyme for 3' hydroxylation and greatly increases cyanidin production over the wild-type, indicating that this mutation is biochemically significant. The change from glycine to aspartic acid at residue 111 is apparently more a "gain-of-function" mutation to make more cyanidin as much as a "loss-of-function" in terms of delphinidin. This view suggests that 'PL' would have more cyanidin in its petals than 'CP', as the mutant F3'5'H is predicted to act as an F3'H. This is what is observed (Fig. 3b).

Fig. 3.
(B) Pigment profile for 'CP' and 'PL', measured at the S5-S6 stage. Relative abundance is measured as peak area under each identified MS peak.

CybeRose note: This is an interesting demonstration of the change of a single amino acid altering the function of an enzyme. There is one problem, though. In this report it appears that 'Painted Lady' should segregate as a unit character. Experimenters in the 19th century had different results. Capt. Trevor-Clarke, for example, made the indicated cross, then isolated a strain that closely resembled 'Painted Lady', but with wings edged and streaked with blue. Charles Darwin also made the cross, and achieved results that suggested methylation might be involved. Maybe a transposon, rather than a gene mutation.

Capt. Clarke's Blue-edged Sweet Pea