The Plant Journal (2006) 48, 414–426
doi: 10.1111/j.1365-313X.2006.02880.x
AtCAT6, a sink-tissue-localized transporter for essential amino acids in Arabidopsis Ulrich Z. Hammes*,†, Erik Nielsen, Loren A. Honaas, Christopher G. Taylor and Daniel P. Schachtman* Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63122, USA Received 15 May 2006; accepted 7 July 2006. * For correspondence (fax þ1 314 587 1521; e-mail
[email protected] or fax þ49 9131 852 8751; e-mail
[email protected]). † Present address: Molecular Plant Physiology, Friedrich-Alexander University Erlangen-Nu¨rnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Summary Amino acids represent the major form of reduced nitrogen that is transported in plants. Amino acid transporters in plants often show tissue-specific expression patterns and are used by plants to transport these metabolites from source to sink during development and under changing environmental conditions. We identified one amino acid transporter, AtCAT6, which is expressed in sink tissues such as lateral root primordia, flowers and seeds. Additionally AtCAT6 was induced during infestation of roots by the plantparasitic root-knot nematode, Meloidogyne incognita. Quantitative reverse-transcriptase PCR revealed nematode inducibility throughout the duration of nematode infestation and in nematode-induced feeding sites. Promoter analyses confirmed expression in endogenous sink tissues and nematode-induced feeding sites. In Xenopus oocytes, AtCAT6 mediated electrogenic transport of proteinogenic as well as nonproteinogenic amino acids with moderate affinity. AtCAT6 transported large, neutral and cationic amino acids in preference to other amino acids. Knockout mutants of this transporter failed to grow on medium containing L-glutamine as the sole nitrogen source. Our data suggest that AtCAT6 plays a role in supplying amino acids to sink tissues of plants and nematode-induced feeding structures. Keywords: sink tissue, amino acid transporter, nematode, essential amino acid, Meloidogyne incognita, pathogen interaction.
Introduction Amino acids represent the major form of transported organic, reduced nitrogen in most plant species. All proteinogenic amino acids are found in both phloem and xylem with concentrations being higher in the phloem. The amino acids aspartate, glutamate and their respective amides asparagine and glutamine are the predominant amino acids in phloem and xylem (Coruzzi, 2003). Amino acids passively follow the phloem bulk flow, the direction of which is dictated by the major osmolyte, generally sucrose (Mu¨nch, 1930). Therefore sink tissues must efficiently take up amino acids, especially those which are less abundant, to meet the demands of tissue. Amino acid concentrations in the vasculature as well as in specific tissues are dynamic and change in response to abiotic stimuli such as light or drought and in response to seasonal changes (Cyr et al., 1990; Lam et al., 1995; Newton et al., 1986; Salleh and Owen, 1984). Amino acid concentrations also vary in response to pathogens and parasites such as aphids and nematodes (Krauthausen and Wyss, 1982; 414
Salleh and Owen, 1984; Sandstrom et al., 2000). Sedentary plant-parasitic nematodes, such as cyst and root-knot nematodes induce highly specialized feeding sites in their host plants. These nematodes derive their supply of essential amino acids by withdrawing them from their host through highly specialized feeding structures which function as strong terminal sinks (Jones and Northcote, 1972; McClure, 1977). Nematode-induced sink tissues consist of cellular structures that share similarities with specialized transfer cells in which transport processes are highly upregulated (Bird, 1961; Jones and Northcote, 1972; Offler et al., 2003). Transfer cells or similar cellular structures that contribute to the amino acid supply of sink tissues are typically found in seed coats or the tapetum (Lee and Tegeder, 2004; Tegeder et al., 2000) and in plant tissues which are involved in nutrient flow during parasitic and symbiotic interactions of plants. In order to respond to numerous physiological needs and environmental changes, plants have evolved several amino acid transporters with distinct but overlapping expression ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd
Nematode-inducible amino acid transporter 415 patterns and physiological properties (Lalonde et al., 2004). The majority of putative amino acid transporter ‘genes fall into two superfamilies: the amino acid transporter (ATF1) superfamily (41 members) and the amino acid-polyaminecholine (APC) superfamily (14 members) (Wipf et al., 2002). Most of the amino acid transporters from plants that have been characterized belong to the ATF1 superfamily, with the amino acid permease (AAP) family being the best studied subfamily among them (Boorer and Fischer, 1997; Boorer et al., 1996; Fischer et al., 1995, 2002; Okumoto et al., 2002). APC transporters from plants are only poorly understood. In plants APC transporters of the L-type amino acid transporter (LAT) subfamily (five members) have not been characterized and only a few members of the cationic amino acid transporters (CAT) family (nine members) have been studied (Frommer et al., 1995; Su et al., 2004). The CAT transporters contain 11 to 14 putative membrane-spanning domains and localize to the plasma membrane or the vacuole. The amino acid transporters from the ATF and APC families in plants are proton-coupled high-affinity transporters (Lalonde et al., 2004). Except for the proline transporters, all amino acid transporters display a broad substrate specificity (Rentsch et al., 1996). In this study we describe the isolation and characterization of AtCAT6, a sink-specific amino acid transporter, the expression of which is induced by nematodes. Results Expression analysis of the AtCAT6 gene AtCAT6 is a member of the APC family of amino acid transporters with a molecular weight of about 65 kDa and contains 11–14 trans-membrane domains (Schwacke et al., 2003; Su et al., 2004). The expression of this gene was found to be induced during infestation of Arabidopsis roots with the root-knot nematode, Meloidogyne incognita. Real-time RT-PCR was carried out to determine AtCAT6 transcript
abundance in response to nematode infestation and in various organs of Arabidopsis (Figure 1). In roots of infested plants, AtCAT6 expression levels were higher 1, 2 and 4 weeks after infestation than in control plants of the same age. The difference between expression levels in infested and uninfested plants increased over time (Figure 1a). The expression levels in manually dissected root-knots 2 weeks after infestation were more than threefold higher than in the rest of the infested root, indicating that the increase of AtCAT6 expression observed in whole roots is mainly due to elevated expression in root-knots (Figure 1b). In soil-grown, uninfested wild-type plants expression levels were highest in fully opened and mature flowers (Figure 1c). Expression levels in the mature flowers were two times higher than those in young, unopened flower buds. AtCAT6 is also expressed in siliques throughout development. The lowest levels of expression were detected in leaves and stems, and expression was barely detectable in roots. Expression of AtCAT6 in sink tissues To obtain more detailed information about the tissue specificity of AtCAT6 expression, we conducted a reporter gene study in which the AtCAT6 promoter was used to drive bglucuronidase (GUS) expression. A total of 30 individual lines containing the PAtCAT6:GUS constructs were screened for b-glucuronidase activity. The staining pattern was consistent in 27 independent transgenic lines. The GUS expression patterns described below are consistent with patterns measured by real-time RT-PCR from various plant tissues and with data in two expression databases: AMPL (http://www.cbs.umn.edu/arabidopsis/) and GENEVESTIGATOR (http://www.genevestigator.ethz.ch/) (Ward, 2001; Zimmermann et al., 2004). These results indicate that all the elements necessary for proper expression are contained in the 1817 bp promoter fragment and that AtCAT6 is active in sink tissues.
Figure 1. Expression of AtCAT6 during nematode infestation and in various tissues determined by quantitative real-time RT-PCR. (a) Transcript abundance in roots 1, 2 and 4 weeks after nematode infestation (open bars) and control plants at the same age (closed bars). (b) Transcript abundance in manually excised knots and in ‘knot-free’ root sections of infested plants 2 weeks after infestation. (c) Transcript abundance in various tissues of soil-grown wild-type (Col-0) plants. Young flowers were not yet opened, old flowers were opened. Young siliques were 0.5 cm.
ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 414–426
416 Ulrich Z. Hammes et al.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Figure 2. AtCAT6 promoter activity in PAtCAT6: uidA promoter–reporter gene plants visualized by GUS staining. (a) Root-knot approximately 2 weeks after infestation. Knots are frequently associated with lateral roots (arrow) and in some occasions adventitious lateral roots are formed (arrowhead) which show intense staining. (b) Section through a root-knot 2 weeks after infestation at a similar developmental stage as shown in (a), the giant cells are stained (arrows). In flowers (c) promoter activity is detected in petals and anthers. In siliques (d) staining is visible in the vasculature, the parenchyma and the developing seeds. (e) Seedling 1 day after germination and (f) seedling 2 days after germination; seed and aleurone layer (arrowhead) are stained. Throughout root development (g–i) promoter activity is detectable exclusively in the root tip: (g) lateral root meristem induction, (h) lateral root meristem formation and (i) root tip.
(i)
In nematode-infested roots GUS staining was visible in root-knots (Figure 2a). In thin sections through root-knots, the promoter activity was strongest in nematode-induced giant cells (Figure 2b, arrows). In uninfested, soil-grown plants strong GUS staining was observed in flowers, predominantly in anthers, particularly in pollen, and to a lesser extent in petals (Figure 2c). During silique development staining was observed in the vasculature of the silique, the funiculus and the developing seeds (Figure 2d). One day after germination weak staining was observed in the entire seedling with the most intense staining in the root tip (Figure 2e). Two days after germination weak staining was observed in the cotyledons and strong staining in the root tips of the seedlings (Figure 2f). Staining was absent from the hypocotyl. Staining was also observed in the endosperm layer (Figure 2f, arrowhead), indicating a possible role for AtCAT6 in providing amino acids to the seed and redistribution of amino acids within
the developing seedling. In roots staining preceded the formation of the lateral root meristem and was visible in the primordia of lateral roots and in the root tips of the lateral roots (Figure 2g–i). A specific staining pattern was not observed in the stems or in fully expanded leaves. After an extended period of incubation in X-Gluc, faint staining was observed in the parenchyma of young leaves and stems. In all cases staining was absent from the vasculature of those tissues (data not shown). Taken together these results show that AtCAT6 expression mainly localizes to plant tissues that are thought to be amino acid sink tissues. Functional characterization of AtCAT6 in Xenopus laevis oocytes To examine the transport characteristics of AtCAT6, the Xenopus laevis oocyte expression system was used.
ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 414–426
Nematode-inducible amino acid transporter 417 Figure 3. The addition of L-alanine to bath solutions induces currents in oocytes expressing AtCAT6 but not in water-injected oocytes. Current traces were obtained from an AtCAT6 cRNA-injected oocyte (a, b) and a water-injected oocyte (c, d) before (a, c) and after (b, d) the addition of 10 mM L-alanine to the bath solution ([Hþ] ¼ 3.2 lM). (e) The voltage pulse protocol was from 40 to )160 mV in 20 mV increments. (f) L-alanine-induced currents were obtained for single oocytes by subtracting the steady-state currents in the absence of substrate from the Lalanine-induced currents, plotted as a function of Vm: L-alanine 50 mM ( ), 20 mM ( ), 10 mM ( ), 2 mM ( ) and 0.5 mM (d).
Oocytes injected with AtCAT6 cRNA accumulated significantly (P < 0.01) more radiolabelled [35S]-L-methionine than water-injected control oocytes. AtCAT6-expressing oocytes accumulated 30.1 4.6 pmol L-methionine per oocyte more than water-injected oocytes after a 15-min incubation (n ¼ 5 oocytes; [Hþ] ¼ 3.2 lM). This showed that AtCAT6 is a functional amino acid transporter in Xenopus oocytes. To further investigate the biophysical properties of this transporter, oocytes expressing AtCAT6 were bathed in 10 mM L-alanine and recorded using a two-electrode voltage clamp system (Figure 3). L-alanine induced inward currents in oocytes expressing AtCAT6, indicating that amino acid transport through AtCAT6 is electrogenic. The substrate-induced inward currents were fully reversible and returned to baseline levels after removal of L-alanine from
the oocyte bathing solution. Recordings from a representative experiment with an AtCAT6-expressing oocyte in the absence (Figure 3a) and in the presence (Figure 3b) of Lalanine illustrate the substrate-induced currents. Currents were induced in AtCAT6-expressing oocytes while no currents were induced upon the introduction of L-alanine to the bath solution containing the water-injected oocytes (Figure 3c,d). Oocytes injected with AtCAT6 displayed a background current in the absence of substrate (Figure 3a). This current was probably due to the movement of protons since it was strongly dependent on the pH of the bath solution and decreased at more alkaline pH values (not shown). These recordings were obtained by stepping the membrane potential from the holding potential, )40 mV, to ranges between )160 and 40 mV in 20-mV increments (Figure 3e).
ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 414–426
418 Ulrich Z. Hammes et al. The current traces consisted of an initial capacitive transient, which relaxed to a steady-state level about 50 msec after the onset of the voltage pulse. At membrane voltages more negative than )80 mV, the currents displayed some time-dependent features and reached steady-state levels 150 msec after the onset of the voltage pulse. Steady-state alanine-dependent currents recorded between )160 and 40 mV displayed a supralinear dependence on voltage, did not saturate at negative potentials and did not reverse at positive potentials (Figure 3f). The same response was observed for all neutral amino acids. In contrast steady-state currents obtained for both positively and negatively charged amino acids saturated between )120 and )160 mV (not shown). Substrate selectivity To investigate the substrate specificity of AtCAT6, bath solutions containing oocytes were perfused with various substrates at a concentration of 2 mM at [Hþ] ¼ 3.2 lM (Figure 4). With the exception of L-proline, all proteinogenic amino acids induced steady-state currents in oocytes expressing AtCAT6 at a membrane voltage of )160 mV. The addition of amino acids to bath solutions containing water-injected oocytes did not induce currents greater than 5 nA (not shown). AtCAT6 transported large and uncharged amino acids in preference to other amino acids (Figure 4). The non-proteinogenic amino acid L-norvaline induced currents similar to proteinogenic amino acids with the same properties, indicating that the large non-polar side chain contributes to substrate recognition. Compared with amino acids with large and uncharged side groups, amino acids with polar side chains, positively charged side chains or small side chains induced smaller currents in oocytes expressing AtCAT6. The anionic amino acids L-glutamate and L-aspartate induced less current than their respective amides, indicating that their negatively charged side chains are responsible for their poor substrate recognition. Very small amino acids such as L-glycine and c-amino butyric acid (GABA) were barely transported by AtCAT6. The small dipeptide L-alanylalanine did not induce currents in oocytes injected with AtCAT6 cRNA, demonstrating that this peptide, and perhaps others, are not transported by AtCAT6. D-alanine induced significantly (P < 0.01) smaller currents in AtCAT6-expressing oocytes as compared to L-alanine, indicating that AtCAT6 exhibits stereo-specific substrate recognition. In the context of the pathogen inducibility of AtCAT6, it is noteworthy that at physiological pH, some of the best substrates for this transporter, as determined by the magnitude of induced currents, are six out of the eight essential amino acids for animals (L-leucine, L-phenylalanine, L-methionine, L-isoleucine, L-tryptophan, L-valine).
Figure 4. Substrate selectivity of AtCAT6. Steady-state currents induced by various substrates (2 mM) were recorded at )160 mV and [Hþ] ¼ 3.2 lM. Substrate-induced currents from water-injected oocytes were typically
