Isolation and characterisation of genes involved in regulation of vacuolar pH and their effect on flower colour

• Research unit: EU RFP
• Project number: 97.0570

Key words (English)

Flower colour; pH genes; pH regulation; vacuole

Alternative project number (English)

EU project number: BIO4CT980432

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Further information (English)

Full name of research-institution/enterprise:
Universität Zürich
Institut für Pflanzenbiologie
Labor für molekulare Pflanzenphysiologie

Partners and International Organizations (English)

Coordinator: Dr. M. Akerboom, Florigene Europe (NL)

Abstract (English)

1.Investigation of the role of pH genes on proton fluxes

In order to know wether altered vacuolar pH is due to altered expression of the vacuolar pumps we performed
expression analysis of several Petunia pH mutants. RNAs was isolated from wild type Petunia and pH mutants. Since
isolation of large amounts of RNA from flowers is a time consuming task we established a fiable method for
quantitative RT-PCR. These experiments showed that no detectable differences were observed between the wild-type
plants and the pH mutants for the transcript levels of the A- and B-subunit of the vacuolar H+-ATPase. Western blots
with microsomes isolated from wild-type and mutant Petunia confirmed these results on the protein level. Similarly, no
differences between the mutant and control plants were observed for the vacuolar H+-PPase. Our results indicate that
the proton pumps are not the primary target for pH genes in Petunia. In order to investigate whether the ATP regulated
channel of the vacuolar membrane corresponds to a proton channel we performed experiments using intact vacuoles and
radiolabelled methylamine, a compound enabling to measure the vacuolar pH very accurately. The experiments didn't
show a difference between vacuoles incubated in the presence of MgATP and ATP alone, indicating that the vacuolar
ATP-modulated channel does not exhibit a high permeability for protons.

2.Uptake and accumulation of flavonoids and anthocyanins

Saponarin, a barley flavonoid is taken up by a proton antiport mechanism in isolated barley mesophyll vacuoles.
Interestingly, mesophyll vacuoles isolated from a barley mutant containing only trace amounts of flavonoids exhibit a
strongly reduced uptake activity. Arabidopsis does not synthesize saponarin. Nevertheless, vacuoles isolated from
Arabidopsis cell cultures take up saponarin. However, in contrast to barley vacuoles, uptake does not depend on the
electrochemical gradient since bafilomycin A1, a potent inhibitor of the vacuolar H+-ATPase and NH4+, which dissipate the
proton gradient, are not inhibitory. These results together with the observation that vanadate efficiently inhibits saponarin
uptake, suggests that in Arabidopsis an ABC-type transporter is involved in vacuolar saponarin transport. These results may
be important for the modification of flower colors, since it shows that flavonoids not synthezied in a plant are nevertheless
taken up into the vacuole.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 97.0570