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First published online August 24, 2007; 10.1104/pp.107.101824 Plant Physiology 145:478-490 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Flavonoids Are Differentially Taken Up and Transported Long Distances in Arabidopsis1,[W],[OA]Genomic Interactions Group, Australian Research Council Centre of Excellence for Integrative Legume Research, Research School of Biological Sciences, The Australian National University, Canberra, Australian Capital Territory 0200, Australia (C.S.B., M.A.D.); and Biology Department, Wake Forest University, Winston-Salem, North Carolina 27109 (G.K.M.)
Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by time-course fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H+-ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.
Flavonoids are plant secondary metabolites that have important developmental and physiological functions (Taylor and Grotewold, 2005  ). Flavonoids are implicated in the control of auxin transport (Jacobs and Rubery, 1988; Mathesius et al., 1998; Murphy et al., 2000; Brown et al., 2001; Buer and Muday, 2004; Peer et al., 2004), allelopathy (Bais et al., 2004), pollen function in some species (Coe et al., 1981; Mo et al., 1992; Taylor and Jorgensen, 1992), signaling to symbiotic microorganisms (Redmond et al., 1986; Djordjevic et al., 1987; Harrison, 2005; Wasson et al., 2006), somatic embryogenesis (Imin et al., 2006), UV protection (Li et al., 1993), defense (for review, see Treutter, 2005), flower coloring (Mol et al., 1998), and seed dispersal (Debeaujon et al., 2000), and appear to accumulate in progenitor cells of root organs (Morris and Djordjevic, 2006). The biosynthetic pathway is known and several mutants with defects in genes encoding these enzymes occur in Arabidopsis (Arabidopsis thaliana; Fig. 1
). Flavonoids are likely synthesized by a cytoplasmic metabolic complex (Winkel-Shirley, 2001), and the expression of genes encoding flavonoid biosynthetic enzymes is regulated developmentally (Winkel-Shirley, 2002) and by light (Pelletier and Shirley, 1996; Kubasek et al., 1998; Jenkins et al., 2001).
Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue (for review, see Waller and Nowacki, 1978 ). More recent grafting experiments showed that root-synthesized metabolites, perhaps carotenoids, regulate shoot development (Sorefan et al., 2003; Van Norman et al., 2004). The current view is that flavonoids are synthesized in the cells in which they accumulate and serve local functions (Peer et al., 2001). Flavonoids are also sequestered in vacuoles or secreted to the environment (Grotewold, 2004). Several studies hypothesized that flavonoids may move from their site of synthesis (Djordjevic et al., 1997; Saslowsky and Winkel-Shirley, 2001; Buer and Muday, 2004). For example, light is required for flavonoid synthesis in Arabidopsis seedlings (Pelletier and Shirley, 1996; Kubasek et al., 1998; Jenkins et al., 2001), and, when seedlings are grown with roots exposed to light, the presence of flavonoids regulates root branching (Brown et al., 2001) and gravitropism (Buer and Muday, 2004). Yet, for these regulatory roles of flavonoids to be relevant when plants are grown in light with roots in the dark, it may be necessary for either light signals or flavonoids themselves be transported to the roots. Therefore, flavonoid movement could have important physiological functions.
Fluorescence microscopy of seedlings stained with diphenylboric acid 2-aminoethyl ester (DPBA; fluoresces upon binding flavonoids) provides a powerful tool to examine the accumulation, localization, and movement of flavonoids in living plants (Sheahan et al., 1998
The Arabidopsis tt4 mutants are helpful in demonstrating the function of flavonoids using the mutant phenotype and chemical complementation with flavonoid precursors. Multiple tt4 alleles have altered auxin transport and auxin-dependent physiological processes, such as gravity responses and root development (Brown et al., 2001 To test the hypothesis that flavonoids move long distances in Arabidopsis, we applied flavonoid aglycones locally to various tissues of the tt4 mutants. We found that DPBA staining combined with epifluorescence and confocal microscopy provided a sensitive and versatile method to follow flavonoid entry, conversion to downstream compounds, and rapid movement from the localized application sites. We used confocal microscopy to examine which tissues harbored the DPBA fluorescence and determined which tissues facilitated long-distance flavonoid movement. To verify that flavonoids were capable of root-to-shoot and shoot-to-root movement, we used HPLC analysis of tissues distal to the application site to show that downstream products of the flavonoid pathway were present in these tissues. Finally, to determine how this movement was facilitated, we tested various inhibitors of ATP-binding cassette (ABC) transporters and H+-ATPase and described a putative mechanism for this movement. Together, these results showed that flavonoids can move in Arabidopsis and suggest that this movement may be mediated by an ABC-type transporter.
A Physiological Necessity for a Flavonoid Movement System
Etiolated wild-type Arabidopsis seedlings accumulate no flavonoids in roots (Buer and Muday, 2004
DPBA Binds to Flavonoids, Generating Intense Fluorescence in Vitro and in Planta
DPBA is a molecule that binds to flavonoids and fluoresces in vitro and in vivo. The specificity of this dye for flavonoids was previously demonstrated, as tt4 mutants make no flavonoids and have no fluorescence upon DPBA treatment (Sheahan and Rechnitz, 1993
To verify this observation in planta, aglycones were supplied to tt7 and tt3 mutant root tips, which have lesions in various genes encoding the flavonoid pathway enzymes (Fig. 1; Shirley et al., 1995
Exogenous Aglycone Applications to tt4 Root Tips Revealed Rapid Movement to Distal Tissues To provide temporal and spatial information on how flavonoids move within plants, two alleles of the flavonoid-deficient tt4 mutant seedlings [tt4(2YY6) and tt4-1] were treated with the aglycones N, DHK, DHQ, K, and Q. These compounds were applied to the root tip in an agar cylinder also containing DPBA, and fluorescence micrographs were captured every 15 s (Fig. 3, bottom). The position of flavonoid fluorescence at 0, 3, 6, and 9 min postapplication is shown. These images were combined into movies showing the fluorescence as a function of time and are available online as Supplemental Movies S1 to S5. N, DHK, and DHQ were not visualized until converted to downstream flavonoid products, and the major product formed was consistent with the fluorescence from Q. The accumulation of Q from these precursors was not present at the root tip, but was restricted to the distal elongation zone and more basal positions. DPBA staining was detected as far as 1 mm from the root tip after 10 min. In contrast, K was taken up and fluoresced green in combination with DPBA at the root tip and was converted to Q after movement away from the tip. This reaction was slower than when applying N, DHK, or DHQ. The K images were collected every 1 min, and the final image was taken 25 min postapplication. Finally, bright golden Q fluorescence was evident in the agar and Q was taken up, resulting in fluorescence at the root tip after 10 min. The overnight application of flavonoid compounds to the root tip resulted in DPBA fluorescence in the leaf tissues from all compounds except K and Q (not shown). In separate experiments, we also examined the movement of flavonoid intermediates, when DPBA was applied uniformly to tt4 seedlings by incubation in a solution containing this dye. This analysis was performed with microscopy at 5 and 60 min after aglycone application, and the subsequent distal fluorescence accumulation is shown in Supplemental Figure S2. Measurements were obtained for the flavonoid movement with this method of visualization to determine the distance of movement after 1 h, and these results are presented in Table I (under basipetal movement).
Control experiments with solvents, Suc, and other chemicals used in the aglycone applications were tested for possible effects on flavonoid movement. The presence or absence of these compounds did not significantly change the experimental outcomes (Supplemental Table S1). In particular, the similar movement of flavonoids in the absence of Suc suggested that flavonoid movement was not the result of Suc-driven loading.
To test the direction of flavonoid movement after localized applications to different sites, we applied the aglycones 5 mm above the root tip (termed midroot) and measured the distance that flavonoid fluorescence moved. N, DHK, and DHQ only moved toward the root tip (acropetal direction). There was no significant differences between N, DHK, and DHQ movements in either tt4-1 or tt4(2YY6) within these categories. We did not find evidence for acropetal or basipetal movement of K and Q when applied midroot. We rarely detected florescence in this tissue upon K or Q application, and in no cases was flavonoid fluorescence found at locations distant from the point of application, consistent with limited uptake and movement of K and Q. The overnight application of N, DHK, and DHQ to the cotyledons of Arabidopsis tt4 seedlings resulted in DPBA fluorescence in the root/shoot junction, midroot, and at the root tip (rsj, mr, and rt, respectively; Fig. 4 ). As with midroot applications, K and Q applications did not result in flavonoid movement from the application site. These results support the idea that precursors to K and Q probably are the molecules that move with conversion to K and Q occurring at the sites of DPBA fluorescence.
HPLC Analyses Confirmed Flavonoid Compounds Are in Tissues Distal to Application Sites
The accumulation of K and Q in tissues distal to the application of N treatment was confirmed using qualitative and quantitative HPLC. N was applied either to the root tip or to the cotyledons for 24 h and extracts from roots and aerial tissues were analyzed. The HPLC profiles of the aglycone standards were compared to crude extracts from untreated tt4-1, tt4-1 treated with N, and wild-type root and shoot extracts. These extracts were analyzed directly and after hydrolysis to remove any glycosides from flavonoid compounds, and representative chromatograms are presented in Figure 5
. The absorption spectra maxima are shown in Supplemental Figure S3 and summarized in Supplemental Table S1, and selected spectra are compared to eluted peaks in Figure 5, a to c. A series of injections was also made using K as an internal standard. These elution times remained consistent with the crude extracts and hydrolyzed samples, confirming the quality of the chromatography. The control tt4-1 mutant had no detectable flavonoid compounds as reported previously (Fig. 5A; Burbulis et al., 1996
To quantitatively determine flavonoid compound amounts in the exogenously fed seedlings compared to the wild type, standard curves were generated using serially diluted authentic flavonoid standards and these results are presented in Table II . The wild type had higher concentrations of the glycosylated compounds than found in N-treated tt4-1 extracts as quantified in the hydrolyzed extract. This difference may reflect the very local application, the small amounts of flavonoids that are taken up, or the limited time for flavonoid synthesis after N treatment in tt4-1 compared to the wild type. N was not detected in any of our assays, suggesting immediate conversion to downstream products or the presence of extremely low concentrations. These results indicated that local application of N to the root tip or cotyledons of tt4-1 led to the accumulation of downstream flavonoids in distant locations in the plant.
Flavonoids Localized in Tissues Where Auxin Transport Occurs and Moved Symplastically Confocal laser scanning microscopy (CLSM) was used to differentiate the tissue and cellular localization of flavonoid compounds after the exogenous application of flavonoid pathway intermediates (Fig. 6 ). In general, DPBA fluorescence localized in the epidermal and cortical cell layers when compounds were applied at the root tip (Fig. 6, A, C, E, G, and H), consistent with the localization of the fluorescence in the videos (Supplemental Movies S1–S5). In contrast, when N, DHK, and DHQ were applied midroot, DPBA fluorescence was detected in the vascular tissue (Fig. 6, B, D, and F). DHQ application led to the most widely distributed DPBA fluorescence pattern, with fluorescence found across the entire root, although the brightest fluorescence was in the epidermal layer. All the other compounds tended to localize specifically in the outer cell layers after root tip applications. These localization patterns are summarized in Supplemental Table S2.
To determine if the transported flavonoids localized apoplastically or symplastically, 0.4 M NaCl was added to the mounting medium to induce slight plasmolysis. The results after applying N or DHQ at the root tip are shown in Figure 7 . Plasmolysis suggested the flavonoid fluorescence was localized within cells, as the fluorescence retracted from the cell walls within the plasma membrane. Similar results occurred with the other exogenously applied aglycones (not shown). If the flavonoid compounds were localized at the plasma membrane, the fluorescence would be brighter at the cell edges owing to the stacking in the z-direction of fluorescence down the side of the cell. The fluorescence in this case was uniform across the cell, supporting intracellular localization and cell-to-cell movement via the symplasm.
Glybenclamide and Glutathione Inhibited Flavonoid Movement
ABC transporters are implicated in the movement of plant secondary metabolites into vacuoles (Yazaki, 2006
Flavonoids Move to Sites Distal of Aglycone Application in Tissues That Transport Auxin
These experiments used two approaches to show that flavonoids move from their site of synthesis or local application to distant tissues. First, flavonoids are not made in roots of etiolated wild-type seedlings (Buer and Muday, 2004 The addition of N, DHK, or DHQ resulted in the uptake, rapid conversion to downstream products, and the long-range movement of downstream products, irrespective of application site. The local application of N, DHK, or DHQ to various tissue types bypassed the chalcone synthase lesion in tt4, resulting in the rapid production (<5 min) of intracellular gold fluorescence likely resulting from the Q-DPBA complex. Additionally, the rapid movement of either flavonoid precursors or downstream products to distal locations in the plant was detected using DPBA. No differences in the flavonoid movement were noted whether DPBA was added via the agar cylinder (Fig. 3) or used after flavonoid application as in conventional staining procedures (Supplemental Fig. S2). HPLC analysis confirmed the formation of downstream flavonoid compounds in distal tissues after exogenous application of N, DHK, or DHQ to root tips or cotyledons of tt4-1. K and Q application to root tips resulted in limited basipetal movement to approximately 10 mm after 24-h incubation. When K and Q were applied either midroot or to the cotyledons, fluorescence was never observed at sites distant from these application sites. The more limited mobility of K and Q in all tissues suggests that N, DHK, and DHQ are more likely the mobile flavonoids that are then detected at distant sites after conversion to K or Q, which generate the most intense fluorescence products when bound to DPBA. Additionally, plasmolysis experiments combined with confocal microscopy showed that the flavonoid products accumulated inside cells and were not detected in regions between cells, suggesting that the long-distance movement of these molecules was symplastic.
Flavonoid movement approximated the rates and direction of auxin (indole-3-butyric acid and indole-3-acetic acid [IAA]) transport (8–10 mm h–1; Rashotte et al., 2003
The movement of flavonoids should be considered in the context of physiological processes that are modulated by these molecules. Consistent with elevated IAA transport, the flavonoid-deficient tt4 mutants have enhanced lateral root formation (Brown et al., 2001
Two issues required consideration relating to the sensitivity and specificity of DPBA staining. The limit of detection for DPBA-enhanced flavonoid fluorescence in vivo is not known. However, our maximum dose of aglycone was in the order of 50 ng per plant, assuming total uptake from the flavonoid supplied in the agar cylinder at the point of application. This result implied high sensitivity of fluorescence microscopy at detecting the DPBA-flavonoid complex in situ. In addition, the application to root tips of agar cylinders with lower flavonoid concentrations (as low as 1 x 10–9 M for 1 h) was also able to cause gold fluorescence to form in the root tips, consistent with the Q-DPBA complex (not shown). Several lines of evidence suggested that DPBA is specific for intermediates of the flavonoid biosynthetic pathway. Mutants that make no flavonoids, such as tt4, exhibit no DPBA fluorescence (Peer et al., 2001
The 24-h incubation of tt4 mutants with N resulted in golden fluorescence indicative of Q formation; however, HPLC analysis showed the relative concentration of Q was only 2 to 60 ng mg–1 fresh weight after hydrolysis. The small quantities measured indicate the sensitivity of the DPBA fluorescence techniques for visualizing the presence of flavonoid compounds, in particular K and Q. The observation that adding N at 1 nM resulted in detectable fluorescence at the root tip after 1-h incubations (not shown) supported the sensitivity of fluorescence detection, as this concentration is well below the detection limits of Q and K by HPLC (100 nM; not shown). Our measured quantities compared favorably with a previous report of K and Q concentrations in Arabidopsis (Peer et al., 2001
The ability of glybenclamide, GSH, and GSSG to inhibit the movement of flavonoids represents a step toward determining the mechanism by which flavonoids are transported. These results suggest that flavonoid movement may be mediated by ABCC proteins, which are ABC transporters, likely to mediate active transport. Active movement is also supported by the tissue-specific localization of flavonoids, their cell-to-cell movement, and their unidirectional movement when applied midroot. If the movement is diffusion mediated, the flavonoid distribution should be less localized and movement bidirectional.
Interestingly, ABC transporters are also implicated in auxin transport (Geisler et al., 2005
The interaction of GSH and GSSG may reflect issues of redox involving F3'H (TT7; Schoenbohm et al., 2000
The interconversion of flavonoid intermediates as examined in vivo followed the predicted biosynthetic pathway of flavonoid compounds with one exception. The unexpected result was that after treatment with K, the formation of Q was obvious (Fig. 2; Supplemental Fig. S2K; Supplemental Movie S4). According to the standard pathway (Schoenbohm et al., 2000
In conclusion, these results showed that flavonoids are capable of long-distance movement throughout Arabidopsis plants. As flavonoids are important for lateral root development (Brown et al., 2001
Chemicals Chemicals were obtained from Sigma or TransMIT.
The Arabidopsis flavonoid mutants tt3-1 and tt7-1 were obtained from the Arabidopsis Biological Resource Centre (The Ohio State University, Columbus, Ohio). These mutants plus tt4(W85) (now known as tt4-1), tt4(2YY6), tt4-2 [generated by backcrossing tt4(2YY6) to remove a second unlinked mutation, max4; Bennett et al., 2006
The various aglycones were locally applied at the root tip, midroot, or on the cotyledons using agar cylinders as described previously for tritiated auxin applications (Rashotte et al., 2000 Two methods of local flavonoid application were used. For generating movies and testing inhibitors, DPBA was added to the agar cylinder with the various aglycones and seedlings were visualized horizontally. To compensate for this addition, extra agar was added to the MES:Suc portion to make a 1% final agar concentration. A slide was coated with molten control media or inhibitors by pipetting the agar directly onto it. Once the agar cooled, a seedling was moved to the agar and placed on the microscope. To visualize flavonoid fluorescence generation and movement, movies were created from individual micrographs taken at specific intervals following flavonoid application at the root tip. A bright-field image was captured and then an image under 488-nm excitation. The agar cylinder with the desired compound was carefully moved near the root tip and individual images were captured over time. Generally there was a slight gap between the root tip and the agar cylinder. To normalize the time for compounds to travel over this gap, a regression line was generated to determine the average time required (not shown). This time was subtracted from the overall time to obtain the true zero. To minimize photobleaching, the excitation shutter was closed between micrograph captures. Due to the nature of the microscopy, the incubation of seedlings with flavonoid compounds required the seedling to lie horizontally versus the vertical orientation as for the other experiments. Images were obtained every 15 s (except for K, every minute). A final bright-field image was captured, and a montage of the entire root tip region was assembled to allow visualization of the overall fluorescence. After adding the time to each image in PhotoShop, the images were joined into QuickTime format (**.mov) using Apple's iMovie3 software. As the epifluorescent microscope is an inverted style, imaging was performed through the slide and agar. For all other experiments, the DPBA staining was done following the incubation of the various aglycones. The agar cylinders were applied at the extreme tip of the root and the seedlings were then oriented vertically in holders for the duration of the incubation. For midroot and cotyledon application, the seedlings were rotated approximately 120° in the vertical plane to minimize possible capillarity where the compounds could flow down the sides of tissues.
Seedlings were transplanted to petri dishes containing normal Murashige and Skoog media plus the inhibitor of choice and incubated vertically in the light for 24 h. The seedlings were then moved to slides coated with the same concentration of inhibitor and viewed under the microscope as for generating movies. N plus DPBA was supplied at the root tip and the movement of flavonoids was followed over 10 min. The results were compared to the control using equal or unequal variance two-tailed Student's t tests determined from F-test analysis of variance in Excel. At least six replicates were in each category.
Fluorescence intensities of N, DHK, DHQ, K, and Q were tested with a spectrofluorometer (FluorMax-3; Jobin Yvon Horiba) with excitation and emission slit widths at 5 nm. Individual compounds at 10 µM were conjugated with DPBA (0.25% plus 0.0005% Triton X-100) and excited at 488 nm, and the emission spectra were collected from 490 to 800 nm. The results were plotted in Origin 7.0, and the calculus integration option was used to integrate the areas under the curves using zero as the baseline.
Epifluorescence microscopy was performed on an inverted Zeiss Axiovert 200M microscope equipped with P.A.L.M. robotics (Millennium Science). Fluorescence excitation and emission wavelengths were with standard FITC filters with excitation from 450 to 490 nm and an emission long-pass filter from 510 nm. A Hitachi HV-D30 digital camera was used to capture epifluorescent micrographs. Measurements were made using the inbuilt measuring capabilities of the P.A.L.M. system. The aglycones were applied at the root tip or 5 mm from the root tip (termed midroot) and incubated in the light for the required time, and the distance of fluorescence was measured from the local application site after DPBA staining. Confocal microscopy was performed on a Leica TCS SP2 laser scanning microscope (Leica Microsystems). Fluorescence was excited by an argon laser (Enterprise II-651; Coherent) with the 488-nm line and with an emission filter from 510 to 580 nm. The aglycones were applied locally at the desired location, incubated for approximately 1 h, and DPBA stained, and optical sections were collected from half the root diameter. Photobleaching becomes a problem when trying to section through entire roots and the distal half of the root becomes increasingly nonfluorescent.
N (100 µM) was applied to Arabidopsis seedlings on day 5 following seeding in 15-cm petri dishes. For the data in Figure 5, the compounds were applied locally at the root tip and incubated vertically for 24 h, and then the seedlings were excised at the root/shoot junction. The aerial tissue was immediately frozen with liquid nitrogen and pulverized with a mortar and pestle. For the samples in Table III, N was either applied at the root tip or on the leaves. After 24-h treatment, the roots and aerial tissues were harvested separately and immediately frozen with liquid nitrogen. For each 100 mg of fresh tissue, 300 µL of cold acetone was added, and the mixture was vortexed, sonicated for 5 min, and then centrifuged at 16,100g to separate the crude extract from the tissue. The supernatant was used for analysis and the hydrolysis of aglycones from the glycosylated flavonoid compounds. The glycosylated flavonoids from crude extracts were hydrolyzed following the protocol of Burbulis et al. (1996) HPLC analysis was performed on crude extracts or the hydrolyzed samples from wild-type and tt4-1 seedlings on a Shimadzu LC-10 VP Series HPLC with a C18 column (Alltima; 250 x 4.6 mm i.d.). The column was injected with 40 µL from the various treatments and eluted with an acetonitrile plus 1% acetic acid gradient from 10% to 40% (35 min) and then from 40% to 88% (10 min). The column eluent was monitored for absorbance by diode array (190–800 nm). The phenolic standards used were N, DHK, DHQ, K, and Q.
The following materials are available in the online version of this article.
We thank Daryl Webb, Australian National University Electron Microscopy Unit, for help with CLSM. Julie Christie and Riccardo Natoli are thanked for help with the P.A.L.M. system. Paul Ahn from Shimadzu and Charles Horcart, Research School of Biological Sciences (RSBS) Mass Spec Facility, are thanked for assistance with the HPLC. Jeremy Weinman, Genomic Interactions Group, RSBS, is thanked for help with the video clips. Warwick Hillier from the Biomolecular Spectroscopy Facility, RSBS, is thanked for his help with the spectrofluorometer. The valuable comments of Barry Rolfe are appreciated. Received June 18, 2007; accepted August 10, 2007; published August 24, 2007.
1 This work was supported by the Australian Research Council Centre of Excellence for Integrative Legume Research (project no. CEO348212) and the Biotechnology Resource Centre (Australian National University; to M.A.D.), and by the U.S. Department of Agriculture (grant no. 2006–03406 to G.K.M.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Charles S. Buer (charles.buer{at}anu.edu.au).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.101824 * Corresponding author; e-mail charles.buer{at}anu.edu.au.
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), or epidermis (arrows). Scale bars = 50 µm.
