1 / 1

FERTILITY Fertility of the mutant is affected in several ways:

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.

tex
Download Presentation

FERTILITY Fertility of the mutant is affected in several ways:

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 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. • FFT (At4g25640) BIOINFORMATICS • 488-amino acid protein • multidrug and toxin efflux (MATE) transporter • most similar protein in BLAST searches is unknown Vitis vinifera protein and a tomato putative anthocynanin permease (1) • 12 transmembrane spans • 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 • TRANSCRIPT • 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. • FERTILITY • 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. Hydropathy plot of the FFT amino acid sequence predicts a typical MATE protein’s 12 transmembrane spans. 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. 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. DISCUSSION 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-3-ols 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. GROWTH AND DEVELOPMENT 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. Col0 Mutant 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. 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.

More Related