We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT’s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologues within the large Arabidopsis MATE family

Beverley J. Glover

Elinor P. Thompson

Hruz T

Julia M. Davies

English

PubMed

Thompson, Elinor P.

Davies, Julia M.

Glover, Beverley J.

Greenwich Academic Literature Archive

Greenwich Academic Literature Archive (GALA)– the University of Greenwich open access repositoryhttp://gala.gre.ac.uk__________________________________________________________________________________________Citation for published version:Thompson, Elinor P., Davies, Julia M. and Glover, Beverley J. (2010) Identifying the transporters of different flavonoids in plants. Plant Signaling & Behavior, 5 (7). pp. 860-863. ISSN 1559-2316 (print), 1559-2324 (online)Publisher’s version available at:http://dx.doi.org/10.4161/psb.5.7.11894__________________________________________________________________________________________Please note  that  where  the  full  text  version provided on GALA is  not  the  final  published version, the version made available will be the most up-to-date full-text (post-print) version as provided by the author(s).  Where possible, or if citing, it is recommended that the publisher’s  (definitive) version be consulted to ensure any subsequent changes to the text are noted.Citation for this version held on GALA:Thompson, Elinor P., Davies, Julia M. and Glover, Beverley J. (2010) Identifying the transporters of different flavonoids in plants. London: Greenwich Academic Literature Archive.Available at: http://gala.gre.ac.uk/4229/__________________________________________________________________________________________Contact: gala@gre.ac.ukPlant Signaling & Behavior 5:7, 860-863; July 2010; © 2010 Landes Bioscience articLe addendum860 Plant Signaling & Behavior Volume 5 issue 7Key words: auxin, flavonoid, guard cell, pollen, transporterSubmitted: 03/22/10Accepted: 03/23/10Previously published online: www.landesbioscience.com/journals/psb/article/11894*Correspondence to: Elinor P. Thompson; Email: et250@cam.ac.ukAddendum to: Thompson E, Wilkins C, Demidchik V, Davies J, Glover BJ. An Arabidopsis lavonoid transporter is required for anther dehiscence and pollen development. J Exp Bot 2010; 61:439–51; PMID: 19995827; DOI: 10.1093/jxb/erp312.We recently identified a new compo-nent of flavonoid transport path-ways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seed-ling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT’s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologues within the large Arabidopsis MATE family.Plant flavonoids are becoming notorious for their wide and expanding range of possible functions. Beyond UV protec-tion (itself not entirely without debate), further roles have been added in plant development; nodulation and interactions with pathogens; fertilisation; and auxin transport. For such a well-described bio-chemical network, it interesting that few aspects of flavonoid function are clear-cut: perhaps it is the recently established link with auxin, so intimately involved in every aspect of plant development, that consigns them to multiple incompletely-known regulatory pathways. Knowledge is lack-ing, in particular, about the transport of flavonoids. Such transport is necessary1 and we now know that selective uptake of flavonoids and movement of flavonoids through the plant occur.2,3 When narin-genin, dihydrokaempferol and dihydro-quercetin were added to the Arabidopsis tt4 mutant [lacking the enzyme chalcone synthase (CHS) and thus all flavonoids] at root tip, mid-root or to cotyledons, they were converted to downstream products. Grafting on flavonoid-producing tissues to tt4 could also complement the mutation.3Kitamura4 and Buer et al.5 speculate that MATE transporters are good can-didates to enable flavonoid transport at the membrane, allowing the necessary movement from one membrane system to another. The link between MATE pro-teins and flavonoid transport is justified by work in tomato6 and confirmed by the dis-covery of TT12.7,8 Also conforming to this premise is our recent work on FFT (Flower Flavonoid Transporter), a MATE protein probably situated in the tonoplast mem-brane that has a role in flavonoid transport in specialised guard cells and anthers.9Auxin and FlavonoidsThe phenotype of the fft-1 mutant adds to the numerous recent descriptions of fla-vonoids influencing auxin transport. The mutant grew faster than wild type in all conditions we tested. Since Arabidopsis seedlings are very small the difference is subtle but was unvarying and statistically significant up to about 2 weeks post-ger-mination, and culminated in fft-1 flow-ering earlier than wild type. The most notable effects of the fft-1 mutation are floral, namely lack of anther dehiscence and reduced pollen viability, but even these cannot be entirely dissociated from an auxin phenotype.10We do not have exhaustive data on the substrate for FFT, but LC-MS compar-ing fft-1 mutant and Col-0 A. thaliana showed significant changes in the level of a kaempferol diglucoside in some floral tissues, among a range of perturbed flavo-nol glycosides. Although FFT promoter-GUS reporter plants showed strong, specific staining in root tissues, the faster root growth of seedlings remains unex-plained, since we did not find differences Identifying the transporters of different flavonoids in plantsElinor P. Thompson,* Julia M. Davies and Beverley J. GloverDepartment of Plant Sciences; University of Cambridge; Cambridge, UKwww.landesbioscience.com Plant Signaling & Behavior 861articLe addendum articLe addendumadjust to alterations in the types and pro-portions of flavonoids present.HomologuesThe MATE family of proteins clearly transport diverse compounds, from the organic cation tetraethylammonium by a human family member, antibiotics by prokaryotic forms, through to citrate secretion by FRD3 in Arabidopsis.14 Now more whole-genome sequences are being completed, we can see homologues of FFT in many other plants. The most closely related proteins currently seen in plants are strikingly similar, the most similar of which are a 75% identical Ricinus commu-nis putative multidrug resistance protein (EEF49069); the 72% identical Solanum lycopersicum putative anthocyanin perme-ase discussed by Matthews et al. (2003) Only low numbers of seeds are produced in siliques on the primary inflorescence. The importance of flavonoids for pollen development and fertility in maize and petunia is well known.11,12 In contrast, in a surprising paper in 1996, Ylstra et al. showed that the tt4 mutant was fer-tile, with normal pollen development.13 Arabidopsis has been seen since as a rare exception among the flowering plants, in being able to produce viable pollen in the apparent absence of flavonoids. Reduced fertility in fft-1 contradicts the received wisdom on flavonoids in Arabidopsis to some degree, in suggesting that perturba-tion of flavonoid metabolism is certainly capable of interfering with the plant’s fer-tility. It is possible that the solution lies in the detail, with Arabidopsis having mech-anisms to compensate for the absence of flavonoids, but lacking the mechanisms to in flavonoids outside floral tissues in our analyses. Both roots and floral tissues require careful dissection and visualisation to confirm and quantify better the effects on the various kaempferol and quercitin compounds that were altered in fft-1. It would also be interesting to attempt to complement the mutation with a range of flavonoid compounds, and to visualise GUS expression following transformation of fft-1 plants with a dr5 auxin reporter construct.Flavonoids and PollenAs mentioned above, fft-1 plants recov-ered over time, appearing wild type in the mature vegetative phase. Early flowers are profoundly affected by the lesion, how-ever, with anthers frequently nondehiscent and containing little or no viable pollen. Figure 1. (a) Genevestigator developmental expression proiles of FFT (red), at4g00350/DTX34 (blue), at5g38030, DTX30 (green), at1g47530/DTX33 (orange), at3g26590/DTX29 OrFs (purple and yellow). (B) FFt (orange) with atPases at5g57350/ATPase 3 (red), at2g07560/AHA6 (blue) and at1g80660/AHA9 (green).862 Plant Signaling & Behavior Volume 5 issue 7in anthesis, corresponding with the phe-notype of fft-1.Flavonoids in Guard Cells and the AntherA GUS-promoter construct showed that guard cells are the likely principal location of the FFT transcript in aerial tissues. This is not surprising since much of a plant’s phenylpropanoid and flavonoid content is found in the vacuoles of guard cells, which are central to UV protection and environ-mental regulation.18,19 The presence of FFT transcript in the hydathode, anther and nectaries is notable—are special roles required of flavonoids in these spe-cialised guard cells? Of interest here too is the proposal that a partner for the TT12 MATE is the proton pumping-ATPase AHA10 (Arabidopsis H+-ATPase), which creates the gradient for active transport by TT12.20 Notably, there is a similar H+-ATPase, AHA9, that is expressed exclusively in anther tissues.21 Whether this forms an equivalent system in anthers with FFT, like that of TT12/AHA10 in seedcoats, requires investigation.We therefore paired FFT with AHA transcripts in Genevestigator, to find the most similar profile to that of FFT (Fig. expressed in roots. In the Arabidopsis gene expression database (www.arexdb.org17) At1g47530/DTX33 indeed shows very strong expression in the root cortex (Fig. 2).Interestingly, the most similar Arabidopsis MATE to FFT at sequence level, At4g00350/DTX34, has a large peak of expression in pollen, contrasting both with the FFT bioinformatics and our experimental data, neither of which show FFT transcript in pollen. At4g00350/DTX34 is described in TAIR (www.ara-bidopsis.org/index.jsp) as being found in mature and germinated pollen, petal dif-ferentiation and expansion stage, anthe-sis, and the globular stage embryo. In general, bioinformatics data suggest that At4g00350/DTX34 expression resem-bles that of the other close homologue At3g26590/DTX29, although the latter is missing the peak in pollen. These pos-sible transcriptional similarities are inter-esting when thinking of the potential redundancy seen in the fft-1 phenotype and might guide future work. Notably, the expression patterns of many of this group of similar proteins seem to feature and in our paper;9 and a 70% identical Vitis vinifera hypothetical protein. Others are now seen in Populis trichocarpa and Oryza sativa. The most similar A. thali-ana proteins to FFT (DTX35) are 67% identical (At4g00350; DTX34), followed by 51% (At1g47530, DTX33; tagged as a “ripening responsive protein”), 50% (At5g38030, DTX30), and 48% identical (At3g26590, DTX29) family members, but these are part of a large family of 50+ putative proteins, as often seen in this spe-cies (for review, see Yazaki, 2005).15RedundancyAt1g47530/DTX33 and FFT transcrip-tion profiles are similar in collected micro-arrays. The microarray data showing widespread transcription for FFT in the profiling application, Genevestigator,16 correspond with our RT-PCR, namely in the lateral root and root elongation zone; senescent and mature leaves; and floral tissues especially sepal, petal and siliques (Fig. 1). Although alike in many microarrays and throughout development, where FFT has a peak in the young flower, At1g47530/DTX33 peaks in the devel-oping seedling and appears, in this com-puter-based analysis, to be more highly Figure 2. arexdb expression proiles in roots of FFT (at4g25640), at4g00350/DTX34, at5g38030/DTX30, at1g47530/DTX33, at3g26590/DTX29 mate proteins with atPases at5g57350/ATPase 3, at2g07560/AHA6 and at1g80660/AHA9.www.landesbioscience.com Plant Signaling & Behavior 86311. Mo Y, Nagel C, Taylor LP. Biochemical complemen-tation of chalcone synthase mutants defines a role for f lavonols in functional pollen. Proc Natl Acad Sci USA 1992; 89:7213-7.12. Ylstra B, Busscher J, Franken J, Hollman PCH, Mol JNM, van Tunen AJ. Flavonols and fertilization in Petunia hybrid—localization and mode of action during pollen tube growth. Plant J 1994; 6:201-12.13. Ylstra B, Muskens M, van Tunen AJ. Flavonols are not essential for fertilization in Arabidopsis. Plant Mol Biol 1996; 32:1155-8.14. Durrett TP, Gassmann W, Rogers EE. The FRD3-mediated eff lux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiol 2007; 144:197-205.15. Yazaki K. Transporters of secondary metabolites. Curr Opin Plant Biol 2005; 8:301-7.16. Hruz T, Laule O, Szabo G, et al. Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics 2008; 420747.17. Birnbaum K, Shasha DE, Wang JY, et al. A gene expression map of the Arabidopsis root. Science 2003; 302:1956-60.18. Gitz DC, Liu-Gitz L. How do UV photomorphogen-ic responses confer water stress tolerance? Photochem Photobiol 2003; 78:529-34.19. Hutzler P, Fischbach R, Heller W, et al. Tissue local-ization of phenolic compounds in plants by confocal laser scanning microscopy. J Exp Bot 1998; 49:953-65.20. Baxter IR, Young JC, Armstrong G, et al. A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana. Proc Natl Acad Sci USA 2005; 102:2649-54.21. Houlné G, Boutry M. Identification of an Arabidopsis thaliana gene encoding a plasma membrane H+-ATPase whose expression is restricted to anther tissues. Plant J 1994; 5:311-7.22. Lee J-Y, Baum SF, Alvarez J, Patel A, Chitwood DH, Bowman JL. Activation of CRABS CLAW in the nectaries and carpels of Arabidopsis. Plant Cell 2005; 17:25-36.level of control to the complex pathways through which plant metabolites influ-ence developmental programmes.References1. Kubasek WL, Shirley BW, McKillop A, Goodman HM, Briggs W, Ausubel FM. Regulation of f lavonoid biosynthetic genes in germinating Arabidopsis seed-lings. Plant Cell 1992; 4:1229-36.2. Buer CS, Muday GK, Djordjevic MA. Flavonoids are differentially taken up and transported long distances in Arabidopsis. Plant Physiol 2007; 145:478-90.3. Buer CS, Muday GK, Djordjevic MA. Implications of long-distance f lavonoid movement in Arabidopsis thaliana. Plant Signal Behav 2008; 3:415-7.4. Kitamura S. Transport of f lavonoids. In The Science of Flavonoids. Edited by Grotewold E. New York: Springer 2006; 123-46.5. Buer CS, Imin N, Djordjevic MA. Flavonoids: new roles for old molecules. J Integr Plant Biol 2010; 52:98-111.6. Mathews H, Clendennen SK, Caldwell CG, et al. Activation tagging in tomato identifies a transcrip-tional regulator of anthocyanin biosynthesis, modifi-cation and transport. Plant Cell 2003; 15:1689-703.7. Debeaujon I, Peeters AJM, Léon-Kloosterziel KM, Koornneef M. The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug second-ary transporter-like protein required for f lavonoid sequestration in vacuoles of the seed coat endothe-lium. Plant Cell 2001; 13:853-71.8. Marinova K, Pourcel L, Weder B, et al. The Arabidopsis MATE transporter TT12 acts as a vacu-olar f lavonoid/H+-antiporter active in proanthocya-nidin-accumulating cells of the seed coat. Plant Cell 2007; 19:2023-38.9. Thompson E, Wilkins C, Demidchik V, Davies JM, Glover BJ. An Arabidopsis f lavonoid transporter is required for anther dehiscence and pollen develop-ment. J Exp Bot 2010; 61:439-51.10. Cecchetti V, Altamura MM, Falasca G, Costantino P, Cardarelli M. Auxin regulates Arabidopsis anther dehiscence, pollen maturation and filament elonga-tion. Plant Cell 2008; 20:1760-74.1). At1g80660/AHA9 and FFT both dis-play peaks of expression that correspond with development of the young flower, also matched by At5g57350/ATPase 3 and At2g07560/AHA6. Arexdb shows a differ-ent picture in root transcription (Fig. 2).It would be of interest to further anal-yse the temporal expression of FFT in the floral organs to discover any synchroni-sation with anthesis. FFT transcription may also be affected by transcription fac-tors such as CRABS CLAW which are required for anther development.22 More technically challenging would be find-ing which flavonoids occur in the anther endothecium (thus concerned with dehis-cence), or the tapetum (i.e., compounds needed for microsporogenesis and pollen development). Any overlap in expression between different MATE proteins may allow redundancy in some cell types but not in others; for example, fft-1’s most pro-found phenotype is in the anther although its transcript is also found in guard cells in the vegetative tissue of young seedlings, where the mutation did not have any obvi-ous effect (besides the subtle changes in germination and growth at this stage).Overall, although much work remains to be done to establish the details of fla-vonoid transport in particular tissues, the transport of specific flavonoids by differ-ent transporters can be seen to add another 

Identifying the transporters of different flavonoids in plants

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Identifying the transporters of different flavonoids in plants

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