Transcript Document

A MATE protein involved in flavonoid transport in Arabidopsis flowers
Elinor Thompson, Beverley Glover and Julia Davies
Department of Plant Sciences, University of Cambridge, Cambridge, UK
Flavonoids aid reproduction by attracting pollinators and dispersers, but in some plants they also have a direct role in fertility. Our work suggests
that a previously uncharacterised MATE-family protein (FFT) transports flavonoids in guard cells of floral organs and, without it, Arabidopsis not
only has perturbed fertility but also altered growth characteristics.
Null mutant fft seedlings grow faster than WT, and seed size and mucilage are also affected by disruption of the gene. SEM and viability
staining of mutant flowers reveal reduced anther dehsicence and a proportion of defective pollen. As some viable pollen is generated there is
reduced fertility, not complete sterility, but siliques are smaller with fewer seeds per silique than in WT. Examination of flavonoid levels by both
spectroscopy and LCMS reveals various changes in buds and siliques, with a significant reduction in one kaempferol glucoside in particular. FFT
transcript can be amplified from most tissues but GUS-FFT-promoter-transformed plants show most intense staining in guard cells (where
flavonoids are often found) of the inflorescence tissues, particularly those of the nectary and anther. Cotyledon guard cells are strongly stained, as
are hydathode guard cells of mature leaves, and the root tip and root elongation zone. Since flavonoids are implicated in regulation of auxin
transport, we conclude not only that the FFT MATE protein is a transporter of the flavonoid biosynthesis pathway that is involved not just in
reproduction but possibly also in growth regulation.
FERTILITY
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488-amino acid protein
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multidrug and toxin efflux (MATE) transporter
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most similar protein in BLAST searches is unknown Vitis vinifera
protein and a tomato putative anthocynanin permease (1)
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12 transmembrane spans
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glutamate residue important for activity in most MATE proteins (2)
found in FFT’s transmembrane domain 7
•
MYB recognition sequences upstream of the FFT start codon
Fertility of the mutant is affected in several ways:
•seed size variance is significantly greater than WT, i.e., it has larger and
smaller seeds although the mean size is similar;
•silique size varies widely;
•a proportion of pollen has abnormal morphology and surface
characteristics; and
•anther dehiscence is reduced.
Seed length (mm)
FFT (At4g25640) BIOINFORMATICS
0.5
mean
minimum
maximum
0.4
0.3
Hydropathy plot of the FFT amino acid sequence predicts a typical MATE
protein’s 12 transmembrane spans.
0.2
0.1
TRANSCRIPT
0.0
Semi-quantitative RT-PCR of FFT shows highest levels of transcript in
floral tissues and siliques. FFT-promoter-GUS transformed plants show
staining in the nectaries and in the guard cells of cotyledons,
hydathodes and inflorescence. The root tips and elongation zone of
young seedlings also stain blue.
RT-PCR of FFT in floral and vegetative tissues. IS, Immature
silique; UB, unopened bud; BS, bolt stem; CL, cauline leaf; ML,
mature rosette leaf; SL, senescent anthocyanin-pigmented
leaf; day2/day5, seedling age. Lower panel L-R. GUS–
promoter-transformed plants: day 4 seedling; cotyledon guard
cells (GC); mature leaf hydathode GC; root tip; developing
lateral root; nectaries; inflorescence; anther GC; silique apex;
close-up of papillae and stigma GC; developing silique;
developing seed in silique.
Variation in seed size (left) and silique production (middle) in fft (R) vs Col0 (L). Right panel, L-R: Col0 (top)
and fft mutant (below) anthers and pollen in Alexander’s viability stain.
PIGMENT LEVELS
LC-MS of flavonoids showed significantly reduced amounts of a
kaempferol diglucoside in mutant buds and the levels of several other
glycosylated flavonols were altered (Table 1). The ratio of fluorescence
from flavonoids/background chlorophyll in anthers was also significantly
reduced in fft vs WT, confirming a reduction in flavonoids. Chlorophyll
levels and other photosynthetic parameters were unchanged.
Table 1. Relative level of flavonol glycosides in mutant versus wild-type (WT) floral tissues.
Peak and tissue type
qRGR bud
qRG bud
qRG immature silique
kGG bud
RkG bud
RkG immature silique
Mutant (as proportion of WT; mean %)
180
65
49
40
70
73
q, Quercitin; R, rhamnoside; G, glucoside; IS, immature silique; k, kaempferol.
DISCUSSION
GROWTH AND DEVELOPMENT
Proportion germinated (%)
Col0
Mutant
100
80
60
40
20
Col0
Mutant
0
24
48
72
96
Time (h) post-stratification
70
Root growth (mm)
The null, single-insertion, homozygous T-DNA mutant
fft (3) phenotype encompasses a range of effects.
Unlike tt flavonoid mutants, seeds appear normally
pigmented but seed mucilage production by imbibing
seeds is irregular. Germination is faster than in WT
but the overall viability of seeds is lower. In the fft
mutant, root growth is significantly faster up to ~2
weeks from germination, a phenotype that is
consistent on ½ MS with 1%, 5% and no sucrose,
under cold stress and in high light. The altered
germination rate does not seem to affect growth
assays since root length is the same in mutant and
WT at day 4 before the differences are seen in root
growth rate.
Mutant vs Col0 1/2 MS
60
50
40
30
20
Col0 mean
Mutant mean
10
0
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Time (days)
Variation in seed mucilage (top), germination (middle) and growth (lower
panels) of fft mutant vs Col0.
Col0
Mutant
REFERENCES
1. Mathews H et al. Plant Cell 2003, 15:1689–1703. 2. Matsumoto T et al. Am J Physiol Cell Physiol 2008, 294:1074-1078. 3. Alonso JM, et al.
Science 2003, 301:653-657. 4. Kitamura S. In The Science of Flavonoids. New York: Springer; 2006:pp123-146. 5. Debeaujon I et al. Plant Cell
2001; 13:853–871. 6. Marinova K et al. Plant Cell 2007, 19:2023–2038. 7. Jaquinod M. Molecular Cellular Proteomics 2007, 6:394–412. 8.
Burbulis IE et al. Plant Cell 1996; 8:1013-1025. 9. Taylor LP, Grotewold E. Curr Opin Plant Biol 2005, 8:317-323. 10. Brown DE et al. Plant Physiol
2001,126:524–535. 11. Buer CS, Muday GK. Plant Cell 2004, 16:1191–1205. 12. Saslowsky D, Winkel-Shirley B. Plant J 2001, 27:37-48.
It has been hypothesised that MATE proteins are involved in flavonoid
transport in Arabidopsis (4) and work has recently confirmed that the
family member TT12 transports anthocyanins and glycosylated flavan-3ols in the seedcoat (5,6). We have found that another MATE protein,
FFT, is necessary for correct accumulation of flavonols in floral tissues.
FFT promoter-GUS staining occurs in inflorescence guard cells and, as
might be expected from the vacuolar location of most flavonoids, FFT
has been reported to be one of the 30 most abundant tonoplast
membrane proteins (7). It is thought that flavonoids are not vital for
fertility in this plant because the tt4 CHS mutant is fertile (8) — although
it does have reduced seed set and pollen tube growth (9). The
phenotype of fft suggests that flavonoids in Arabidopsis are required for
optimal fertility, at least.
A role in growth regulation is much-discussed as flavonoids can
displace synthetic auxin transport inhibitors (10), flavonoid mutants have
altered auxin transport, and flavonoid fluorescence in the root tip alters
following gravity stimulation (11). Correspondingly, FFT-directed GUS
staining was seen in the cortex in the elongation zone, and in the root
tip. Notably, chalcone synthase and isomerase were identified previously
in the epidermal and cortex cells of the elongation zone and at the root
tip (12). A perturbed flavonoid profile in the fft mutant may therefore also
explain its altered growth rate.
ACKNOWELDGEMENTS
We thank T. Burgess, D. Coomes, I. Furner, J. Hibberd, S.A. Johnson, C. Martin, J. Skepper and C. Wilkins for materials, advice and
helpful discussions, and the BBSRC for funding.