At least four mRNAs for oat phytochrome A (phyA)
are present in etiolated oat tissue. The complete amino
acid sequences of two phyA isoforms (A3 and A4) and the
N-terminal amino acid sequence of a third isoform (A5)
were deduced from cDNA sequencing (Hershey et al.,
1985). In the present study, heterogeneity of phyA on a
protein level was studied by tryptic mapping using electrospray
ionization mass-spectrometry (ESIMS). The total tryptic
digest of iodoacetamide-modified phyA was fractionated
by gel filtration chromatography followed by reversed-phase
high-performance liquid chromatography. ESIMS was used
to identify peptides. Amino acid sequences of the peptides
were confirmed or determined by collision-induced dissociation
mass spectrometry (CID MS), MS/MS, or by subdigestion of
the tryptic peptides followed by ESIMS analysis. More than
97% of the phyA3 sequence (1,128 amino acid residues) was
determined in the present study. Mass-spectrometric analysis
of peptides unique to each form showed that phyA purified
from etiolated oat seedling is represented by three isoforms
A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced
changes in phytochrome in vivo phosphorylation site at
Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595–10599)
as well at Ser17 and Ser598 (known as in vitro phosphorylation
sites) were also analyzed. The extent of phosphorylation
at Ser7 appears to be the same for phyA isolated from dark-grown
and red-light illuminated seedlings. In addition to Ser7,
Ser598 was identified as an in vivo phosphorylation site
in oat phyA. Ser598 phosphorylation was found only in phyA
from the red light-treated seedlings, suggesting that the
protein phosphorylation plays a functional role in the
phytochrome A-mediated light-signal transduction.