JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

3-04
Functional Analysis of Flavonoid Accumulation Genes
of Arabidopsis thaliana
S. Kitamura a) , F. Matsuda b) , T. Tohge c) , K. Yonekura-Sakakibara b) , M. Yamazaki c) ,
K. Saito b, c) and I. Narumi a)
a) Radiation-Applied Biology Division, QuBS, JAEA, b) RIKEN PSC, c) Chiba University
Plant cells have the potential to produce thousands of
secondary metabolites, the production of which is finely
regulated by developmental programs and environmental
stimuli for each cell type. Flavonoids are one of the most
widely found secondary metabolites in the plant kingdom,
and show multiple functions in planta such as contributing
to the attractive color of flowers and fruit and protection
against UV-light and microbes. Flavonoids are synthesized
at the cytoplasmic surface of endoplasmic reticulum (ER)
within the cells. On the other hand, the final accumulation
sites of some flavonoid end-products such as anthocyanins
and proanthocyanidins (PAs) are known to be located in the
central vacuole. This suggests that plant cells have
mechanisms by which flavonoids are transported from the
ER (synthesis site) to the vacuole (accumulation site). We
previously isolated ion-beam-induced Arabidopsis mutant
tt19, which showed aberrant accumulation pattern of
anthocyanins and PAs. Physiological analysis indicates
that TT19 gene is involved in intracellular flavonoid
transport. Here, in order to address the flavonoid transport
mechanism more deeply, flavonoid accumulation mutants of
Arabidopsis were analyzed at the cell biological and
metabolomic levels.
TT19 gene was expressed as a fusion protein with GFP.
GFP-TT19 proteins were observed only in anthocyanin- and
PA-accumulating cells, implying a correlation between
TT19 function and flavonoids. Within PA-accumulating
cells, GFP-TT19 proteins were detected in the cytosol.
This suggests that TT19 functions before vacuolar uptake of
flavonoids. Vacuolar uptake of PAs is executed by a
transporter protein TT12. The tt12 mutant could not
accumulate PAs in the vacuoles, whereas the tt19 mutant
could accumulate them in small vacuolar-like structures.
In tt12 tt19 double mutant, PAs were observed outside the
vacuolar-like structures. These results indicate that
formation of the vacuolar-like structures harboring PAs is
TT12-dependent. Given the function of TT12 as a PA
transporter, it is conceivable that the vacuolar-like structures
in tt19 are small “vacuoles”.
We next observed PA-accumulating cells by differential
interference contrast microscopy. Under this observational
condition, tt19 mutant specifically showed unique, thick
spheres. These spheres were not detected in wild-type and
other mutants. Histochemical analysis demonstrated that
distribution pattern of these spheres overlapped the pattern
of PA-accumulating small vacuoles in tt19. These findings
prompted us to speculate that the composition and/or
conformation of the PAs in the vacuoles differed between
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tt19 and wild-type. To investigate this, PAs were analyzed
by acid-hydrolysis and LC-MS. We found higher levels of
solvent-insoluble PAs in tt19 compared with wild-type.
Metabolic profiling of the solvent-soluble fraction by
LC-MS demonstrated that PA derivatives such as
epicatechins and epicatechin oligomers, although highly
accumulated in wild-type, were absent in tt19. We also
revealed that tt12 specifically accumulated glycosylated
epicatechins, the putative transport substrates for TT12.
Based on these metabolic and cytological characteristics
of mutants, we propose a model concerning the PA pathway
(Fig. 1). The Arabidopsis PA precursor epicatechins,
synthesized by anthocyanidin reductase (ANR) in the
cytosol, are at least in part converted to epicatechin-
hexosides by glycosyltransferase (GT). Following the
synthesis of epicatechins and epicatechin-hexosides,
cytosolic TT19 protein functions to protect the chemical
properties of these precursors. Epicatechin-hexosides are
taken up in the vacuoles by transporter proteins such as
TT12, and the hexose moiety is removed by glycosidase in
the vacuoles to polymerize the PA precursors. TT19 is
required for the synthesis of regular PA polymers, but it may
not be essential for the vacuolar accumulation of PA
derivatives via TT12. This model will help to create plants
possessing novel flavonoid constitution.
Fig. 1 A model concerning PA accumulation pathway.
Putative steps are indicated as dotted arrows.
Abbreviations used: ANR, anthocyanidin reductase;
GT, glycosyltransferase; GST, glutathione
S-transferase.