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Plant Molecular Biology 36: 377–385, 1998. c 1998 Kluwer Academic Publishers. Printed in Belgium.

Amino acid carriers of Ricinus communis expressed during seedling development: molecular cloning and expression analysis of two putative amino acid transporters, RcAAP1 and RcAAP2 Julie-Ann Bick, Anil Neelam, J.L. Hall and Lorraine E. Williams School of Biological Sciences, Biomedical Sciences Building, University of Southampton, Southampton SO16 7PX, UK ( author for correspondence, e-mail: [email protected]) Received 25 April 1997; accepted in revised form 16 September 1997

Key words: amino acid transport, carrier, expression analysis, RT-PCR, in situ hybridisation, Ricinus

Abstract This study reports on the isolation of two putative amino acid carrier cDNAs, RcAAP1 and RcAAP2, from Ricinus communis. Northern analysis shows that RcAAP1 and RcAAP2 are expressed abundantly in the cotyledon and root tissues of developing seedlings and at lower levels in the endosperm and hypocotyl. In the mature plant low expression was observed in the source and sink leaves. We have further characterized the expression of RcAAP1 in Ricinus roots by in situ hybridization. The transcripts are localized in many cell types of the root tip region, including the epidermal and cortical cells, but the highest expression was observed in the cells of the stele situated adjacent to the xylem poles. This is the first report describing the cellular expression of an amino acid transporter in roots, and the results are discussed in relation to the physiological role of this transporter. Introduction Plants cannot assimilate elemental nitrogen directly and therefore they have developed several means of ensuring that all tissues recieve a sufficient supply of this essential nutrient. Nitrogen is available in the soil mainly in the form of nitrate, ammonium and amino acids. Although nitrate is the main source of nitrogen to most plants, amino acid uptake becomes more significant for plants that grow in soils low in inorganic nitrogen. The importance of this uptake in the overall nitrogen nutrition varies with different plant species and their particular habitats. In soils that are mineralpoor but rich in organic nitrogen (in the form of amino acids), amino acid transport can account for up to 80% of the total nitrogen uptake [15]. Amino acids may also act as regulators of nitrate uptake into the roots since high concentrations of amino acids supplied by the shoots have been observed to inhibit the uptake of nitrate by the roots [20]. In addition, nitrate uptake can The nucleotide sequence data reported will appear in the EMBL and GenBank Nucleotide Sequence Databases under the accession numbers Z68759 (RcAAP1) and Y11121 (RcAAP2).

be inhibited by the presence of amino acids supplied directly to the roots [1, 6]. As the plant develops, reallocation of nitrogen is necessary to support the growth of other developing tissues (e.g. flowers and seeds). The long-distance translocation of nitrogen, mainly in the form of amino acids, occurs via both the xylem and phloem. Amino acids are transported from the root tissue to the leaves in the xylem, and represent the predominant compounds of the xylem sap. During remobilization of nitrogen, amino acids are translocated in the phloem, and here they are the second-most abundant constituent, after sucrose. There is now a great deal of physiological and biochemical evidence for specific amino acid carriers involved in the transport of different amino acids into and around the plant [2, 36]. An extensive molecular characterisation of amino acid carriers has been carried out in Arabidopsis where evidence has been presented for a multi-gene family of amino acid permeases (AAPs), the members of which differ in their tissue distribution and substrate specificity [7]. The range of carriers observed is probably due to their diverse

378 physiological roles in the plant. For example, in the root, amino acid carriers may be involved in uptake of amino acids from the soil, transfer of amino acids to the xylem for mobilization from the root, unloading of amino acids from the phloem in the root and possibly direct transfer between the phloem and xylem for recycling [25, 26]. In leaves, amino acid carriers are necessary for transfer from mesophyll cells to the apoplast and for uptake from the apoplast into the phloem. There may also be a system for taking up amino acids from the pool delivered in the xylem [9]. In order to determine their physiological role it is now important to identify the range of carriers present in higher plants, to precisely map their distribution, and to determine their biochemical properties. Transport of amino acids into the developing seed is a particularly important process since this is their main source of nitrogen, and there is evidence for a carrier system transporting amino acids out of the maternal tissue and one for uptake into the embryo and/or endosperm in certain species [29]. In addition, amino acid transport is very important during germination to ensure an adequate nitrogen supply to the rapidly growing embryo [23]. Our work on amino acid carriers has concentrated on the systems present in the germinating and developing Ricinus communis seedling, an excellent model system for such studies since it shows high rates of amino acid transport in various organs [23, 25]. Kinetic studies, using isolated membrane vesicles, have indicated the existence of several amino acid carriers in the cotyledons and roots [31, 32, 33, 34, 35, 36]. A molecular description of the amino acid carriers in this species is particularly useful because, unlike Arabidopsis, there are already a great deal of physiological and biochemical data available, not only specifically concerned with amino acid carriers but also for assimilate partitioning and phloem and xylem transport [11, 16, 25, 26, 36]. The present study reports on the isolation of two partial-length cDNA clones, RcAAP1 and RcAAP2, encoding putative amino acid carriers from Ricinus. The organ- and tissue-specific expression of these carriers was investigated, and the results are discussed in relation to the possible function of these transporters in the Ricinus seedling. This is the first report describing the cellular expression of an amino acid carrier (RcAAP1) in root tissue.

Materials and methods Plant material Ricinus communis L. var. sanguineus (castor bean) seeds were imbibed in cold running tap water for 24 h prior to planting in Vermiculite. They were grown in the dark, at 28  C, for 6 days. To obtain mature plants, 4–5-day old seedlings were potted in Levingtons F7 compost, and grown in the temperate greenhouse for about 3 months. Reverse transcriptase polymerase chain reaction (RT-PCR) of total RNA First-strand cDNA was synthesised from total RNA (5 g) isolated from roots of Ricinus using 10 ng of oligo(dT)12,18 with 200 units of Superscript reverse transcriptase (Gibco-BRL, Paisley). This singlestranded cDNA was used as a template in PCR using the primers described below. For each reaction, 20 l of denatured cDNA mixture was added to 10 l of 10 Taq buffer, along with magnesium chloride to a final concentration of 1.5 mmol/l, 1.0 mmol/l dNTPs and 50 pmol of each of the two amino acid carrier primers; the reaction mix volume was made up to 100 l with sterile water, before the addition of 2.5 units of Taq polymerase enzyme (Gibco BRL). The PCR reaction was carried out in a thermal cycler (Hybaid, Teddington) for 40 cycles with a primer annealing temperature of 50  C and an extension temperature of 72  C. Primers for PCR amplification of amino acid carrier sequences The degenerate PCR primers were designed using sequence data from the published Arabidopsis amino acid carriers AAP1 [8, 13] and AAP2 [17]. The forward primer (CAGGAATTCGCG(A/T/C)ATT(C/A)GCG(A/T/C)CAG(A)C(T)TG(A/T/C)TGG) is based on sequence located in the first of twelve transmembrane domains of AAP1 and AAP2, and includes some non-membranous sequence. The reverse primer (CGGGAATTCG(A/T/C)GCG(A)AAG(A/T/C)ACC(T)TGG(A)TAG(A/T/C)GCG(A/T/C)CC) is based on sequence located within the ninth transmembrane domain. Each primer is 30 bp in size, and includes EcoRI sites (underlined) to facilitate the cloning of PCR products.

379 Cloning and sequencing of RT-PCR cDNA products RT-PCR cDNA products were digested with EcoRI and gel-purified products were cloned into the EcoRI site of pGEM 7Zf, plasmid vectors. Both strands of the recombinant clones were sequenced with a Li-Cor automated sequence with IRD-labelled T7 and SP6 promoter primers using the Thermosequenase cycle sequencing kit (Amersham, Little Chalfont). Southern analysis Genomic DNA was isolated from Ricinus cotyledons according to Shure et al. [27]. The DNA was restricted with EcoRI, HindIII and XhoI and blotted onto Hybond N+ membrane (Amersham). The blots were hybridised overnight in a buffer containing 5 SSC, 1% Boehringer blocking agent (Boehringer, Mannheim), 0.1% N -laurosarcosine, 0.02% SDS at 65  C, with 32 P-labelled RcAAP1 and RcAAP2 DNA probes prepared by random priming using a Ready-To-Go DNA labelling kit (Pharmacia, St. Albans). Successive washing of the blots was carried out under conditions of moderate stringency (2 SSC, 0.1% SDS, room temperature; 2 SSC, 0.5% SDS, 45  C; 0:2 SSC, 0.5% SDS, 45  C) and the blots were autoradiographed at ,80  C. Experiments were repeated three times and representative blots are shown. Northern analysis Total RNA was isolated from various Ricinus tissues as described by Logeman et al. [18]. 25 g of each RNA sample were separated on a 1% agarose gel containing formaldehyde, and blotted onto N+ membrane (Amersham). The blots were hybridized with 32 P-labelled RcAAP1 and RcAAP2 cDNA probes at 68  C in a buffer containing 5 SSC, 1% Boehringer blocking agent, 0.1% N -laurosarcosine, 0.02% SDS. After hybridisation the blots were successively washed under conditions of moderate stringency (2 SSC, 0.1% SDS, room temperature; 0:5 SSC 0.1% SDS, 50  C; 0:1 SSC, 0.1% SDS, 55  C). Northern analysis using the Arabidopsis STP1 cDNA probe was carried out under low-stringency conditions at 55  C in a buffer containing 5 SSC, 1% Boehringer blocking agent, 0.1% N -laurosarcosine, 0.02% SDS. The blots were washed under low-stringency conditions (2 SSC, 0.1% SDS, room temperature; 1 SSC, 0.1% SDS, 50  C). Equal loading of RNA samples was confirmed by staining

with ethidium bromide. Experiments were repeated three times and representative blots are shown. In situ hybridisation cRNA probes were produced using the digoxygeninRNA labelling kit (Boehringer) according to the manufacturers’ instructions. Limited hydrolysis of cRNA probes was carried out according to Cox et al. [4]. Fixing, PEG embedding, and tissue pre-treatments including DNase and proteinase K digestion were based on those described by Marrison and Leech [19]. Sections were pre-hybridized in buffer containing 50% formamide, 5 SSC, 2% Boehringer blocker, 0.1% N -laurosarcosine, 0.02% SDS, 50% formamide and 100 ng/ml denatured salmon sperm DNA at 50  C for 6 to 24 h. The buffer was then removed and replaced with hybridization buffer containing 5 SSC, 2% Boehringer blocker, 0.1% N -laurosarcosine, 0.02% SDS, 50% formamide, 100 ng/ml denatured salmon sperm DNA and 600 ng/ml probe RNA of hybridization buffer, at 50  C, for 16 to 36 h in a sealed humid chamber. After hybridisation, the sections were washed in fresh pre-warmed buffer containing 50% formamide, 2 SSC at 50  C for 30 min and then briefly in NTE buffer containing 500 mmol/l sodium chloride, 10 mmol/l Tris-HCl pH 7.5, 1 mmol/l EDTA, pre-warmed to 30  C. Unbound probe RNA was then degraded by treatment with RNase A enzyme, at a concentration of 1000 units/cm3 in buffer containing 10 mmol/l Tris-HCl pH 7.5, 1 mmol/l magnesium chloride, at 30  C for 30 min. The sections were then washed under high-stringency conditions using prewarmed 0:1 SSC, 0.1% SDS, 20% formamide for 30 min at 50  C. Immunological detection was carried out using alkaline-phosphatase-conjugated antidigoxigenin antibodies, based on the manufacturer’s instructions (Boehringer) but including 10% (w/v) PVA in the colorimetric reaction [5]. Experiments were conducted three times and the results shown are from representative sections. Histochemical procedures Phloroglucinol, aniline blue and Sudan III staining procedures were carried out as described by Jensen [14]. Fluorol yellow staining were performed according to Brundrett et al. [3].

380 Sequence comparisons and alignments The Genetics Computer Group programs (University of Wisconsin, USA) were used to search for sequence homology. These are available through the Seqnet facility at Daresbury Laboratory, Daresbury, UK.

Results Molecular identification of amino acid carriers in Ricinus using the polymerase chain reaction (PCR) Degenerate oligonucleotide primers used for the amplification of amino acid carrier sequences were designed based on the published sequences of the amino acid carrier clones, AAP1/Nat 2 and AAP2, isolated from Arabidopsis [8, 13, 17]. Two conserved amino acid motifs in these sequences were used: AIAQLGW which is located in the first of the putative twelve transmembrane domains and includes some non-membranous sequence and GAYQVFA, located within the ninth transmembrane domain. The predicted size of an amplified amino acid carrier sequence using these primers is ca. 860 bp, and such a product was amplified after RT-PCR of Ricinus root RNA. Cloning and sequencing of the 860 bp PCR products resulted in the isolation of two distinct but related cDNA sequences and these are referred to as RcAAP1 and RcAAP2 (Ricinus communis amino acid permease 1 and 2). A comparison of the amino acid sequences of the two Ricinus clones is shown in Figure 1. RcAAP1 and RcAAP2 share 62% identical amino acids. As shown in Table 1, the Ricinus amino acid sequences also show high homology to the protein sequences of the AAP amino acid carriers which have been isolated from Arabidopsis [7]. RcAAP1 showed high sequence homology with AAP2 and AAP3 with 80 and 81% respectively whereas RcAAP2 showed 82% identity with AAP6 [22]. A small gene family of amino acid carriers exists in Ricinus Southern analysis of amino acid carrier genes was carried out using either RcAAP1 or RcAAP2 as probes under stringency conditions which prevent their crosshybridisation. The conditions were determined by DNA dot blot analysis (data not shown). RcAAP1 and RcAAP2 showed distinct patterns of hybridization (Figure 2). Under the moderate-stringency conditions used, several faint bands were revealed in addition to

Figure 1. Predicted amino acid sequence of RcAAP1 (RAAC1) and RcAAP2 (RAAC2) cDNAs (accession numbers Z68759 and Y11121).

Figure 2. Southern blot analysis of the Ricinus amino acid permease clones, RcAAC1 and RcAAP2. Ricinus genomic DNA (50 g) was digested to completion with DNA restriction enzymes as indicated, hybridized with 32 P-labelled RcAAP1 (A) or RcAAP2 (B) cDNA probes. Lane 1, EcoRI; lane 2, XhoI; lane 3, HindIII.

two or three strongly hybridizing ones depending on the restriction enzyme used. This analysis suggests the presence of a family of amino acid carrier genes in Ricinus.

381 Table 1. Sequence identities between Ricinus RcAAPs and Arabidopsis AAPs. The identity between pairs of amino acid transporters is indicated by the percentage of identical amino acids in the sequences.

RcAAP1 RcAAP2 AAP1 AAP2 AAP3 AAP4 AAP5 AAP6

RcAAP1

RcAAP2

AAP1

AAP2

AAP3

AAP4

AAP5

AAP6

100 62 60 80 81 77 71 63

100 75 62 64 63 59 82

100 55 56 57 54 73

100 72 88 66 57

100 72 72 58

100 66 58

100 56

100

RcAAP1 and RcAAP2 show tissue-specific expression The expression of RcAAP1 and RcAAP2 in different tissues of Ricinus was investigated by northern blotting. This analysis identified transcripts for each carrier of ca. 1.9 kb; these were detected in all tissues with variable levels of expression (Figure 3). The highest expression of RcAAP1 and RcAAP2 was observed in the cotyledon with appreciable but lower expression levels in the root. In contrast, markedly lower expression of both RcAAP1 and RcAAP2 was detected in the hypocotyl and endosperm, and in the sink and source leaves of the mature plant. There was little appreciable difference in the levels of expression of either gene in cotyledons from seedlings 3 to 5 days old. Because of the broad similarity in expression profiles observed with RcAAP1 and RcAAP2, we also determined the pattern of hexose carrier gene expression using an STP1 cDNA probe (Figure 3). This encodes a hexose carrier from Arabidopsis [24] and, in this case, the expression pattern was very different with expression confined to the main sink organs. Strongest signals were detected in the root, sink leaf and endosperm with transcript sizes of ca. 2.1 kb; no signal was detected from the cotyledon, hypocotyl or source leaf tissues. Cellular expression of RcAAP1 In situ hybridisation analysis of RcAAP1 in the Ricinus root tip region (0.25–5 cm from tip) was carried out in order to obtain information about the cell specificity of this amino acid carrier in roots. Histochemical analysis was also performed to characterize the structure of the root at this stage of development (results not shown).

Figure 3. Northern analysis of RcAAP1 (RAAC1), RcAAP2 (RAAC2) and STP1 homologues in Ricinus seedling and leaf tissues. Total RNA (25 g) from various Ricinus tissues was hybridised with 32 P-labelled probes. A representative blot is shown for each probe. The figures given under each lane are the mean relative densities of the hybridization signals obtained from three independent northern analyses given as a percentage of the value obtained from the 5-day cotyledon (or root tissue for the STP1 hybridization analysis). Lanes: 1, 2, and 3 are for cotyledons 3, 4 and 5 days after germination respectively; lane 4, root; lane 5, hypocotyl; lane 6, endosperm; lane 7, sink leaf; lane 8, source leaf.

This indicated that the Ricinus root has a fairly standard pattern of organization consisting of an outer epidermis

382 and several layers of cortical cells. The endodermis is evident and the protoxylem has a tetrarch arrangement with the phloem distributed between the xylem poles. The results from in situ analysis after hybridization with either the sense or antisense digoxigenin-labelled cRNA probe is shown together with control sections treated with RNase (Figure 4). The reaction observed in most cells treated with the antisense probe is higher than that observed with the sense probe and, furthermore, the hybridization signal is reduced by an RNase pre-treatment. Cells of the epidermis display a slightly stronger signal than do the cortical cells; however, the strongest hybridization signal is observed from cells located within the stele situated adjacent to each of the xylem poles. In situ hybridization was also carried out with sections from cotyledons 6–7 days old, however it was not possible to define the cellular expression in this case due to the high background signal from this particular tissue (results not shown). Discussion Previous work has indicated that there are several different amino acid carriers present in Ricinus which may be involved in a variety of physiological processes (for a review see [36]). Therefore, we embarked on this study to characterize these transporters at the molecular level using a number of procedures. RT-PCR is a sensitive technique that has been used in the analysis of a number of plant gene families, including that of the hexose carrier genes in Ricinus cotyledons [30] and the family of alcohol dehydrogenase genes in Glycine max [21]. In the present study, cDNA prepared from Ricinus root RNA was subjected to PCR using degenerate oligonucleotide primers designed from published sequence data for amino acid carriers AAP1 and 2 from Arabidopsis [8, 17]. This approach lead to the isolation of two partial-length cDNAs, RcAAP1 and RcAAP2 (Figure 1), which show high homology to the AAP family of amino acid transporters from Arabidopsis (Table 1). This provides strong evidence that RcAAP1 and RcAAP2 clones encode amino acid carriers related to the Arabidopsis AAP family. With the increase in the cloning of membrane transport proteins, a number of gene families encoding functionally related proteins have been identified. These included the plasma membrane H+ -ATPase family of genes from Arabidopsis [12], the vacuolar ATPase gene family [28] and the AAP transporters [7, 8]. In order to determine if a family of amino acid carriers is present in Ricinus, Southern hybrid-

ization with RcAAP1 and RcAAP2 clones was used to analyze the Ricinus genome. This was carried out under conditions of stringency which prevent the crosshybridization of RcAAP1 and RcAAP2. Southern analysis of RcAAP1 and RcAAP2 identified a small number of prominent bands along with several fainter bands. The stronger bands are probably due to the hybridization of RcAAP1 and RcAAP2 with the corresponding genomic sequences whereas the fainter bands may represent the hybridization of these probes with other related genomic sequences. There was no correlation between the RcAAP1 and RcAAP2 Southern hybridization patterns, and it is therefore possible that other genes related to RcAAP1 and RcAAP2 are present in the Ricinus genome indicating the existence of a small gene family. In support of this, we have recently isolated a cDNA encoding a third amino acid carrier, RcAAP3 (Neelam and Williams, unpublished). Members of gene families often exhibit different temporal and tissue-specific expression. Northern blotting has been used to compare the tissuespecific expression of five amino acid carriers (AAP1– 5) cloned from Arabidopsis [7]. This analysis demonstrated the distinct patterns of expression of each gene in the family, covering every organ of the plant. In the present study, the level of expression of RcAAP1 and RcAAP2 was compared in various tissues of the seedling and mature plant. These genes both showed highest levels of expression in the cotyledons, lower levels in the root, but were much less abundant in the other tissues. No marked changes were observed in the expression levels as the cotyledons expanded during day 3–5 of germination. During this period of development, nutrients (mainly sucrose and amino acids) are received solely from the endosperm tissue. Since cotyledons are symplastically isolated from the endosperm, a potential role for such carriers in the cotyledon is in the uptake of amino acids and sucrose from the apoplastic space. A different pattern of expression was observed for homologues of the hexose carrier STP1. Northern analysis using the STP1 probe identified high expression of a homologous transcript in the root, with lower expression in the sink leaf and endosperm tissues. This is consistent with a possible role for this carrier in apoplastic phloem unloading via the retrieval of hexose produced from the hydrolysis of sucrose by an apoplastic invertase. No transcripts homologous to STP1 were detected in the Ricinus cotyledons, hypocotyl and source leaf tissues under these conditions.

383

Figure 4. In situ localization of RcAAP1 mRNA in Ricinus root tip. In situ hybridizations were performed on transverse sections from Ricinus root tip region (first 5 mm) using digoxygenin-labelled antisense or sense cRNA probes, in vitro transcribed from the RcAAP1 cDNA clone. Hybridization with RcAAP1 antisense cRNA probe: 16 magnification (a); 41 (d); 150 (f). Hybridization with RcAAP1 sense cRNA probe: 16 magnification (c). RNase A-treated section hybridized with RcAAP1 antisense cRNA: 16 (b); 41 (e). Section labelling as follows: x, xylem pole; p, pericycle; e, epidermis.





RcAAP1 and RcAAP2 transcripts were fairly abundant in the root of the germinating seedling. Here, these carriers may have several possible functions. These include the uptake of amino acids from the soil, xylem loading, amino acid cycling from the phloem or possibly in the retrieval of amino acids leaked from the vascular tissue. To help resolve this question, the expression of RcAAP1 was studied in more detail in root tip sections using in situ hybridization. In transverse root sections, RcAAP1 transcripts were detected in several cell-types. There appeared to be slightly higher levels in the epidermal cells compared to the cortical cells which is consistent with a role for this carrier in amino acid uptake from the soil. However, the highest expression levels were detected in four regions of cells located at the xylem poles, adjacent to the protoxylem.









The cells appear to have thick walls and histochemical staining of the endodermis with Sudan III and fluoral yellow demonstrated that they are located within the stele. It appears therefore that these cells are found in the region of the pericycle adjacent to the protoxylem poles. In Arabidopsis seedlings analysed by whole-mount in situ hybridization, AAP1 was found in the root [17]. Although the particular cellular location was not demonstrated, it was expressed only in the elongation zone behind the root meristem [17]. In Ricinus roots comparative studies using plasma membrane vesicles have demonstrated lower glutamine uptake activity in the root tip than in the remaining root [31]. Therefore, further studies localizing the transcripts along the mature root are required in order to determine whether

384 RcAAP1 is expressed only in specific zones of the root and to determine whether higher glutamine transport activity coincides with an increase in RcAAP1 expression or the expression of another amino acid transporter gene. The localization of RcAAP1 in the pericycle region adjacent to the xylem poles is clearly significant and by analogy with radish and Arabidopsis, these may be the auxin-responsive cells from which the progenitors of lateral root primordia derive [37]. Interestingly, a very similar pattern of expression has also been observed for the auxin-responsive transcript GH3 in soybean roots [10]. We have observed lateral roots developing from these cells in the pericycle region adjacent to the xylem poles further up the root in Ricinus (Bick and Williams, unpublished). The distribution of RcAAP1 could be consistent with a high amino acid requirement of these cells for the active protein synthesis necessary for the initiation of organogenesis. Alternatively, it is possible that these represent specialized cells which actively accumulate amino acids for subsequent transfer to the xylem. Therefore, further studies monitoring the transcript levels during lateral root development will be important for determining the significance of amino acid carriers during organogenesis, while an ultrastructural study of the cells in the pericycle/xylem pole region may provide further insight into their association with the xylem and their role in amino acid transport.

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Acknowledgements The Arabidopsis hexose carrier cDNA clone STP1, (Sauer et al., 1990) was a kind gift from Dr N. Sauer (N¨urnberg, Germany). We would like to thank Dr J.L. Marrison and Prof. R.M. Leech for helpful advice concerning in situ hybridization.

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