AtCAT6 is expressed in terminal sink tissues
Sink tissues in plants are important for plant survival through the establishment of storage tissues such as roots and seeds. These sink tissues often represent the edible portions of the plant which are also a source of essential human nutrients. Sink tissues depend on the translocation of minerals and carbon from other parts of the plant. In order to ensure the supply of these nutrients, specific long-distance and cellular transport mechanisms are needed to direct and load these nutrients into the sink tissues. In this work we identified and characterized an amino acid transporter, AtCAT6, from Arabidopsis which is specifically expressed in sink tissues including developing seeds, lateral roots and nematode-induced feeding structures. In contrast to the expression of AtCAT6 in several sink tissues, other amino acid transporters that are expressed in sink tissues showed either a very specific expression pattern exclusively in one tissue type (Lee and Tegeder, 2004; Rentsch et al., 1996; Schwacke et al., 1999; Tegeder et al., 2000) or expression in both sink and source tissues (Fischer et al., 1995; Okumoto et al., 2002). AtCAT6 appears to be more broadly expressed in plants, but specifically in sink tissues.
In roots infested by M. incognita, a root-knot nematode, feeding sites are established in which one of the most striking features is large giant cells. These cells are thought to function as sink tissues from which the nematode obtains essential nutrients from the host plant. We have shown that expression of AtCAT6 is upregulated by root-knot nematodes in giant cells, and that AtCAT6 is an amino acid transporter protein. These observations, plus the determination that AtCAT6 is localized at the plasma membrane, are all consistent with the transport of amino acids into the cytoplasm of these giant cells to possibly contribute to the nutrition of the nematode. Giant cells are thought to be symplastically isolated from the surrounding tissue (Huang and Maggenti, 1969; Jones, 1980), and therefore nutrients must be taken up across the giant cell plasma membrane. Consistent with this concept, a recent characterization of transporter gene expression in Arabidopsis roots infested with root-knot nematodes showed that at least 50 putative membrane transporters are regulated during the course of the establishment of nematode feeding sites (Hammes et al., 2005). In a microarray study of plants infested by the beet cyst nematode, Heterodera schachtii, expression analysis also revealed that certain transporters are upregulated (Puthoff et al., 2003). The gene encoding the sucrose transporter AtSUC2 was induced in roots infested with the cyst nematode (Juergensen et al., 2003), but was not found to be expressed in syncytia. In roots infected with the cyst nematode the syncytia were shown to be symplastically connected to the phloem which led to the diffusion of reporter gene product into syncytia (Hoth et al., 2005). The situation is different in case of AtCAT6, because the AtCAT6 promoter is not active in the phloem and therefore the reporter gene product would not have entered the feeding site by diffusion. Thus expression of the reporter gene product in giant cells is more likely to be due to promoter activity in giant cells. However, we cannot rule out that symplastic connections exist in the giant cells induced by the root-knot nematode. Symplastic connections would reduce the need for carrier-mediated transport across the plasma membrane except where carriers are needed for retrieval. AtCAT6 may contribute to the supply of essential amino acids for nematode feeding sites, but is not essential for the establishment of feeding sites since feeding sites can be established in plants where AtCAT6 is knocked out. This finding supports the idea that there is redundancy in amino acid transporter expression or compensatory expression of amino acid transporters in the cat6 knockouts if the nutrient loading into those structures depends on an apoplastic step.
The substrate specificity of AtCAT6
AtCAT6 is one member of a nine-member gene family in plants (Wipf et al., 2002). Previous studies indicated that two members of the AtCAT family, AtCAT1 and AtCAT5, are likely to be proton-driven high-affinity transporters that transport mainly cationic amino acids (Frommer et al., 1995; Su et al., 2004). Our characterization of AtCAT6 shows that this transporter has a high affinity for cationic amino acids and is also likely to be energized by protons. Studies on AtCAT1 and AtCAT5 used a yeast system for functional characterization; whereas we were able to characterize the function of AtCAT6 using the Xenopus oocyte heterologous expression system (Dreyer et al., 1999). Characterization in oocytes allowed for the analysis of the transport activity of AtCAT6 with the full complement of proteinogenic as well as a few non-proteinogenic amino acids.
Based on our results, we found some potential similarities and differences between the function of AtCAT6 and the previously characterized AtCAT1 and AtCAT5. For example, we found that AtCAT6 has a lower affinity for positively charged amino acids than AtCAT1 and AtCAT5. AtCAT1-mediated l-histidine transport with a K0.5 of 35 μm, AtCAT5-mediated l-arginine transport with a K0.5 of 12 μm and AtCAT6-mediated l-lysine transport with a K0.5 of 128 μm. While it is possible that AtCAT6 constitutes an amino acid transport system of lower affinity compared with the other CATs, it is also possible that the differences in affinities arise from the use of different heterologous expression systems (i.e. yeast versus oocytes; Dreyer et al., 1999). For example, the K0.5 of the amino acid transporter AtAAP1 for proline in a yeast expression system was approximately 60 μm (Frommer et al., 1993) whereas the K0.5 for proline in Xenopus oocytes was determined to be 3 mm at a membrane voltage of −150 mV (Boorer et al., 1996). In the yeast system membrane potential is not usually controlled or measured during uptake experiments, and in some cases the affinity of transporters varies with membrane potential (Boorer et al., 1996; Chandran et al., 2003; Yao et al., 2000). Therefore membrane potential may account for observed differences between yeast and oocytes. The K0.5 of AtCAT6 decreases with the membrane voltage and was 38 ± 4 μm at a membrane voltage of −40 mV (data not shown) which is a high membrane potential for yeast, but one that has been previously recorded (Kotyk, 1994). Our results correspond well with results (Frommer et al., 1995; Su et al., 2004) showing that the AtCATs transport cationic amino acids with moderate to high affinity.
In this study we found that large neutral amino acids induced larger currents than cationic amino acids in oocytes expressing AtCAT6. Therefore AtCAT6 may differ in substrate specificity from AtCAT1 and AtCAT5 because in previous studies they have been shown to transport the cationic amino acids l-arginine and l-histidine (Frommer et al., 1995; Su et al., 2004). However, direct comparisons between certain functional characteristics of AtCAT1 or AtCAT5 and AtCAT6 cannot be made because large neutral amino acids were not tested in previous studies, except for valine which efficiently reduced histidine transport through AtCAT1 (Frommer et al., 1995). We tested all amino acids at the same concentration (2 mm) using oocytes. Several amino acids were also tested across a range of concentrations in oocytes expressing AtCAT6. l-lysine was tested as a representative positively charged amino acid and l-methionine as a representative large, neutral amino acid. Although AtCAT6 has a higher affinity for l-lysine, the maximum currents were greater for large and neutral amino acids such as l-methionine. The relative transport efficiencies (i.e. relation of K0.5 to ) for transport of l-lysine and l-methionine are 2.1 and 1.7, respectively, thus making both amino acids equally good substrates. It will be interesting to determine whether large, neutral amino acids are transported by other members of the CAT family in plants or whether this is a unique feature of AtCAT6. It is possible that anionic amino acids are transported in their uncharged form. Further experiments using those substrates at higher pH values will be required to address that issue. To determine which amino acids are transported by AtCAT6 in planta, it will be necessary to know the relative concentrations of amino acids as well as the pH in the apoplast of the tissues in which AtCAT6 is expressed.
High-affinity transport of cationic amino acids has also been reported to be mediated by AtLHT1, a member of the lysine/histidine transporter (LHT) family (Chen and Bush, 1997). A recent paper also reported high-affinity transport of neutral and anionic amino acids and little transport of cationic amino acids by AtLHT2 (Lee and Tegeder, 2004). Full characterization of each amino acid transporter is required to draw conclusions about the functional characteristics of the entire family. In contrast to the CAT and LHT families, much more is known about the substrate specificity for the AAP family in plants. AtAAP1 to AtAAP6 were all investigated in detail in the Xenopus expression system (Boorer and Fischer, 1997; Boorer et al., 1996; Fischer et al., 2002). AtAAP1 to AtAAP5 exhibit a moderate or low affinity for their amino acid substrates, a characteristic they share with AtCAT6. Only AtAAP6 displays affinities for some amino acids in the lower micromolar range (Fischer et al., 2002). AtCAT6 exhibits a higher affinity for l-lysine than any AAP. The highest affinity for l-lysine was determined in AtAAP5 (K0.5 = 500 μm; Boorer and Fischer, 1997). Similar to AtCAT6, AtAAP5 also has a high affinity for l-methionine; however, other large and neutral amino acids were not transported well by AtAAP5. In conclusion, the transport characteristics found for AtCAT6 are unique to what has been reported for other plant amino acid transporters in that only this protein mediates the transport of cationic and large, neutral amino acids with high affinity and in the physiological range of the concentration of these substrates (Zhu et al., 2005).
A physiological role for AtCAT6
Knockout mutants of AtCAT6 did not grow on medium containing 2.5 mm or 5 mm of l-glutamine as the sole nitrogen source. This phenotype may be due to the impaired uptake of l-glutamine and hence the mutants died because of nitrogen deficiency. Uptake of l-glutamine is consistent with our studies showing that oocytes expressing AtCAT6 transport glutamine and that yeast expressing AtCAT6 is more sensitive to the toxic l-glutamine analogue (S)-2-amino-6-diazo-5-oxo-l-norleucine (DON) than the control (Su et al., 2004). However, AtCAT6 is not expressed in regions of the root that are involved in the net uptake of nutrients. The expression of AtCAT6 was found mainly in root tips, which are not involved in translocation of nutrients to the rest of the plant. This favours a model in which l-glutamine would be taken up by another unknown amino acid transporter, and the cat6 knockouts are more likely to be impaired in the translocation and/or retrieval of those amino acids. Further studies will be required to establish the precise role of AtCAT6 in the transport of amino acids into sink tissues.
In summary, the transport properties of AtCAT6 partially overlap with a number of other amino acid transporters of plants but may differ in that only AtCAT6 has been shown to mediate both high-affinity transport of both cationic and large, neutral amino acids. These amino acids are essential for animal nutrition and must be obtained from plants or other sources by heterotrophic organisms. When the agronomically important pathogen M. incognita invades plant roots, a feeding site is induced along with the expression of AtCAT6. Invasion by this pathogen triggers giant cells to form which function as sinks. Specific transport processes are altered (Hammes et al., 2005), including the induction of AtCAT6 which may be involved in transporting nutrients required for nematode development. The increased expression of AtCAT6 and, perhaps, other amino acid transporters, in seeds represent a potential target for engineering seeds with increased essential amino acids such as l-lysine and l-methionine that are often limited in a plant-based diet (Schaafsma, 2000).