Plant Molecular Biology 47: 353–366, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Analysis of barley chloroplast psbD light-responsive promoter elements in
Karen E. Thum
, Minkyun Kim
, Christian Eibl
, Hans-Ulrich Koop
and John E. Mullet
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA (
for correspondence; e-mail firstname.lastname@example.org);
Botanical Institute, Ludwig-Maximilians-Universität, Menzinger
Strasse 67, 80638 Munich, Germany;
Present address: Department of Agricultural Chemistry, Division of Applied
Biology and Chemistry, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea
Received 20 November 2000; accepted in revised form 8 May 2001
Key words: chloroplast gene expression, circadian rhythms, light quality, organelle genome, plastid transformation,
The plastid gene psbD encodes D2, a photosystem II reaction center chlorophyll-binding protein. psbD is tran-
scribed from a conserved chloroplast promoter that is activated by blue, white, or UV-A light. In this study,
various forms of the barley (Hordeum vulgare L.) chloroplast psbD-LRP were fused to the uidA reporter gene
and introduced into the tobacco (Nicotiana tabacum L.) plastid genome through homologous recombination.
Primer extension analysis of transcripts from the psbD-LRP-uidA construct showed that the barley psbD-LRP
was activated in tobacco by blue or white light. Transcription from this construct was also regulated by circadian
cycling indicating that the barley psbD-LRP could respond to light modulated regulatory pathways in tobacco.
Mutation of the psbD-LRP prokaryotic −10 promoter element reduced transcription to very low levels in all light
regimes. In contrast, mutation of a prokaryotic −35 promoter element had no effect on transcription from the
psbD-LRP. Deletion or mutation of an upstream activating element, the AAG-box (−36 to −64), also reduced
transcription from the construct to very low levels. In contrast, deletion of the upstream PGT-box (−71 to −100)
did not alter promoter activation by blue light, or responsiveness to circadian cycling. These in vivo studies conﬁrm
the importance of the psbD-LRP −10 promoter element and AAG-box in light regulation and demonstrate that
these elements are sufﬁcient to mediate circadian cycling of the barley psbD promoter.
The chloroplast D1 and D2 chlorophyll-binding pro-
teins form the reaction center core of Photosystem
II, one of four multi-subunit complexes involved in
photosynthetic electron transport. When plants are
illuminated, D1 and D2 are damaged and must be
degraded and resynthesized in order to maintain Pho-
tosystem II function (Mattoo et al., 1984, 1989; Ohad
et al., 1985; Schuster et al., 1988; Christopher and
Mullet 1994). The need for continued synthesis of
D1 and D2 in mature chloroplasts is satisﬁed in part
through differential accumulation of psbA and psbD
transcripts, which encode D1 and D2 (Mullet and
Klein, 1987; Gamble et al., 1988; Baumgartner et al.,
1993). psbA transcripts are maintained at high levels
in mature chloroplasts through light-stimulated tran-
scription (Klein and Mullet, 1990) and extraordinary
mRNA stability (Klein and Mullet, 1987; Klaff and
Gruissem, 1991; Rapp et al., 1992; Kim et al., 1993).
High levels of psbD transcripts are maintained in ma-
ture chloroplasts primarily through a white- or blue-
light-activated psbD promoter (psbD-light-responsive
promoter; psbD-LRP) (Gamble and Mullet, 1989;
Sexton et al., 1990a; Christopher and Mullet, 1993).
The psbD-LRP is highly conserved in higher plants
although the organization of the psbD operon and sur-