Physiologia Plantarum 2014 © 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Accumulation, selection and covariation of amino acids insieve tube sap of tansy (Tanacetum vulgare) and castor bean(Ricinus communis): evidence for the function of a basicamino acid transporter and the absence of a γ -amino butyricacid transporterSusanne N. Bauera, Heike Nowaka, Frank Kellera, Jose Kallarackala,†, Mohamad-Reza Hajirezaeib

and Ewald Komora,∗

aPflanzenphysiologie, Universitat Bayreuth, 95440 Bayreuth, GermanybPhysiologie und Zellbiologie, Leibniz Institut IPK, 06466 Gatersleben, Germany

Correspondence*Corresponding author,e-mail: ewald.komor@uni-bayreuth.de

Received 24 October 2013;revised 9 December 2013

doi:10.1111/ppl.12153

Sieve tube sap was obtained from Tanacetum by aphid stylectomy andfrom Ricinus after apical bud decapitation. The amino acids in sieve tubesap were analyzed and compared with those from leaves. Arginine andlysine accumulated in the sieve tube sap of Tanacetum more than 10-fold compared to the leaf extracts and they were, together with asparagineand serine, preferably selected into the sieve tube sap, whereas glycine,methionine/tryptophan and γ -amino butyric acid were partially or completelyexcluded. The two basic amino acids also showed a close covariation in sievetube sap. The acidic amino acids also grouped together, but antagonistic tothe other amino acids. The accumulation ratios between sieve tube sapand leaf extracts were smaller in Ricinus than in Tanacetum. Arginine,histidine, lysine and glutamine were enriched and preferentially loadedinto the phloem, together with isoleucine and valine. In contrast, glycineand methionine/tryptophan were partially and γ -amino butyric acid almostcompletely excluded from sieve tube sap. The covariation analysis groupedarginine together with several neutral amino acids. The acidic amino acidswere loaded under competition with neutral amino acids. It is concludedfrom comparison with the substrate specificities of already characterizedplant amino acid transporters, that an AtCAT1-like transporter functions inphloem loading of basic amino acids, whereas a transporter like AtGAT1 isabsent in phloem. Although Tanacetum and Ricinus have different minor veinarchitecture, their phloem loading specificities for amino acids are relativelysimilar.

Introduction

Phloem loading of amino acids is a crucial processto supply reduced N to growing tips, flowers andfruits (Pate 1980). The amino acid concentrationsin the sieve tubes range from 40 to more than 600

†Present address: Sustainable Forest Management Division, Kerala Forest Research Institute, Peechi 680653, Thrissur, Kerala,India

mM (Riens et al. 1991, Girousse et al. 1996, Lohaus andMoellers 2000, Tilsner et al. 2005) and can be similarto sucrose concentrations in apoplastic loaders such asMedicago sativa (Girousse et al. 1991). There was noor only a small concentration gradient from cytosol to

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the sieve tube sap, but a steep uphill gradient fromapoplast to sieve tube sap in barley and spinach (Lohauset al. 1995). The amino acid spectrum was rather similarbetween cytosol, apoplast and sieve tube sap, whichwas interpreted that all amino acids were loaded fromthe cytosol via the apoplast into the phloem by broad-spectrum amino acid transporters.

In meantime several dozens of amino acid transportershave been cloned (the majority from Arabidopsis) and,partly, kinetically characterized in heterologous expres-sion systems (reviewed in Fischer et al. 1998, Tegederand Rentsch 2010, Tegeder and Ward 2012). None ofthe transporters was specific for only one amino acidand none of the amino acids was transported by onlyone transport system. Few of identified transporters werepredominantly present in source leaves and, possibly,located in the leaf veins. Their function for phloemloading was suspected, but still speculative (Fischeret al. 1998, Toufighi et al. 2005, Brady et al. 2007,Tegeder and Ward 2012). In addition, as pointed out byFischer et al. (1998), the substrate affinities as revealedin heterologous expression systems may be overruledby the actual concentrations at the sites from where thephloem loading occurs, thus their role in loading of aparticular amino acid is uncertain. The transporters andtheir localization were determined nearly exclusively inArabidopsis thaliana, where data on sieve tube sap com-position are lacking, on the other hand, the compositionof sieve tube sap is known for some plants (Dinant et al.2010), whose amino acid transporters are unknown.

We had obtained many samples of sieve tube sap fromTanacetum vulgare and Ricinus communis. We usedthese samples and: (1) compared the concentrations ofthe amino acids in sieve tube sap and leaf extracts, (2)determined, whether certain amino acids were positivelyor negatively selected from the leaf into the sieve tubesap and (3) tested, if there is a covariation of certainamino acids in the sieve tube sap.

The plant species Tanacetum and Ricinus belong totwo different types of phloem loaders (Gamalei 1989).Tanacetum is an apoplastic phloem loader (group 2b)without symplastic connections between the sievetube-companion cell complex and other phloem cells.It has strong wall ingrowths of the intermediate cellstoward the inner side of the minor veins. Ricinus is amixed symplastic/apoplastic loader (group 1–2a). Thereare some symplastic connections from intermediate cellstoward the companion cell-sieve tube complex andthe cross-sectional area of cell wall and plasmalemmaof the intermediate cells toward the inner side of theminor veins is relatively small. It was interesting toreveal, whether these different types of phloem loading

architecture led to different features of amino acidselection and accumulation in phloem loading.

Materials and methods

Plant material and growth conditions

Tansy plants (Tanacetum vulgare) were grown fromrhizomes in the greenhouse at defined ammoniumnitrate solutions (3 mM) as described by Nowak andKomor (2010). Then they were transferred to a chamberwith 18:6-h L : D cycle at 24/20◦C temperature and60–70% air humidity. Some plants which were used forcovariation analysis were fertilized with 1, 6 or 12 mMammonium nitrate.

Ricinus plants [Ricinus communis (L.) var. sanguineus]were grown in the greenhouse at 25/20◦C day/nightregime and 50% humidity as described in Kallarackalet al. (2012). In one experimental series the Ricinusplants were transferred to 4 or 10 l pots with vermiculate,continuously percolated with fertilizer solution of 1, 3,6 or 12 mM NH4NO3 concentrations and grown inclimate chamber at 26/20◦C day/night regime. Samplesfrom these plants were used for covariation analysis only.

Sampling of leaves

Leaves from 37 tansy plants, which were also used forstylectomy, were collected, frozen in liquid nitrogen andextracted in 80% acetone (Nowak and Komor 2010).

Ricinus leaves were obtained from 40 plants whichwere used for the bleeding sap sampling, too. The leaveswere immediately frozen at −80◦C, lyophilized andextracted with 80% ethanol.

Phloem sap sampling

Phloem sap from tansy was collected by stylectomy onplants of 3–4 months age (Nowak and Komor 2010)in a chamber at 60–70% air humidity and 24◦C roomtemperature. The aphids Uroleucon tanaceti (Mordv.),Macrosiphoniella tanacetaria (Kalt.) or Macrosiphumeuphorbiae (Thomas) were settled on leaves for half aday. When the EPG indicated that the stylet was insertedinto the sieve tube, the stylet was cut by radiofrequencymicrocautery and the exuding sieve tube sap wascollected with 0.5 μl capillaries. The exudation lastedfor a few hours up to 5 days. The capillaries were eitherfrozen for further analysis or emptied into 10 μl distilledwater in reaction tubes and then frozen until analysis.Phloem sap was collected from stylets of 18 plants bytaking two to eight phloem sap samples from each plant.

Eighteen Ricinus plants of 6–8 weeks of age were usedfor bleeding sap collection. The apical inflorescence bud

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of Ricinus was cut (Kallarackal and Milburn 1984) andtwo to six samples of the bleeding sap were collectedwith 20–50 μl capillaries. The first 5 μl after cutting werediscarded. The sap was frozen for further analysis.

Analysis of amino acids

Amino acids were determined by RP-HPLC afterderivatization with ortho-phthaldialdehyde (Nowak andKomor 2010) or derivatization by 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate according to Zurbriggenet al. (2009). The first method did not separatemethionine from tryptophan. Proline mostly remainedundetected because of a low detection limit.

Statistical analysis

The data were analyzed for statistical significance withthe statistics function of SIGMAPLOT 10 (Systat SoftwareInc., San Jose, CA). The covariation of amino acids wasanalyzed with the statistical software PRIMER6 (PRIMER-ELtd., Plymouth, UK). First the data (i.e. the amino acidspecies of each sample) were normalized to yield therelative contribution of each amino acid in each sample.Then the resemblance function Pearson Correlation wasapplied to the data and a hierarchical cluster analysiswas applied. The results were graphically presented inthe cluster mode ‘group average’.

Results

Amino acid concentration range of phloem sapand leaves

The amino acid concentration (amino acids areabbreviated with the common three-letter code) inTanacetum phloem sap varied between 212 and 1457mM. Asp, glu, ser and arg were the major amino acidswith 10–20% each. The amino acid concentration ofTanacetum leaves varied between 1 and 45 μmol g−1

FW with glu, asp, ser and gln as the major amino acidswith 10–20% each.

The amino acid concentration in bleeding sap fromRicinus varied between 15 and 190 mM. Gln was byfar the major amino acid with about 50%, glu and aspwere the next frequent with 5–7% each. The aminoacid concentration in Ricinus leaves varied between 7and 96 μmol g−1 FW, glu, gln, asp and gaba made up10–20% each.

Accumulation, selection and covariation of aminoacids in the sieve tube sap of Tanacetum

Sieve tube sap and leaf extracts were obtained fromplants with 3 mM ammonium nitrate fertilization. Nearly

Table 1. Concentration of amino acids in sieve tube sap and leaf cellwater of Tanacetum. Mean ± SD, significance ***P < 0.001, **P < 0.01,*P < 0.05, t < 0.1. n sieve tube sap = 47, n leaf = 17.

Sieve tubesap (mM)

Leaf (μmol ml−1

leaf water)Ratio (sieve

tube sap/leaf)

ala 6.67 ± 3.69 4.43 ± 1.23 1.51*

arg 20.40 ± 13.22 1.06 ± 1.6 19.2***

asn 12.90 ± 7.81 8.90 ± 12.43 1.45t

asp 28.47 ± 13.38 12.54 ± 8.33 2.27***

gaba 0.10 ± 0.35 1.56 ± 0.91 0.064***

gln 16.75 ± 12.86 9.21 ± 5.99 1.82*

glu 36.74 ± 13.67 10.99 ± 5.18 3.34***

gly 5.66 ± 2.77 1.40 ± 0.74 4.04***

ile 3.65 ± 2.35 1.11 ± 0.74 3.28***

leu 2.93 ± 2.85 0.99 ± 0.85 2.97*

lys 6.70 ± 5.89 0.33 ± 0.35 20.62***

met/trp 2.19 ± 1.52 2.21 ± 1.71 0.99phe 3.89 ± 2.38 1.15 ± 0.54 3.38***

ser 65.59 ± 45.88 8.15 ± 4.51 8.05***

thr 12.09 ± 5.47 4.21 ± 2.69 2.87***

tyr 2.16 ± 1.77 0.55 ± 0.31 3.93**

val 4.85 ± 3.21 2.14 ± 0.95 2.27**

all amino acids were accumulated in the sieve tube sap,arg and lys more than 10-fold, asp, glu, gly, ile, leu, phe,ser, thr, tyr and val 2–8-fold (Table 1). The other aminoacids were only marginally or not accumulated and gabawas almost excluded. A similar picture derived, whenthe relative distribution (i.e. percentage) of amino acidspecies in sieve tube sap and leaves was compared. Argand lys, less so asn, asp and ser, were preferably selectedinto the phloem, whereas met/trp, gly and especiallygaba were strongly negatively selected (Table 2).

The cluster analysis by the Pearson Correlation statis-tics identified a covariation of arg and lys together andseparate from the other amino acids. (Fig. 1A, Table S1).The neutral amino acids (except asn) clustered in threegroups, gly, gln and ala formed one group, the branched-chain together with the phenolic amino acids anothergroup and met/trp with ser the third (Fig. 1A). Asn andgaba were separate from each other, and glu plus asp cor-related negatively to the other amino acids (Fig. 1A), i.e.they decreased when the other amino acids increased.

The grouping of amino acids in the leaves was differentfrom that of sieve tube sap, but not according to thebiochemical origin of the carbon skeletons. Asp andglu clustered together, also ser and gly, and both groupswere slightly reciprocal to the other amino acids (Fig. 1B,Table S2).

Accumulation, selection and covariation of aminoacids in the sieve tube sap of Ricinus

The accumulation ratio of amino acids in sieve tube sapof Ricinus was much lower than in Tanacetum. Arg, gln,

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Table 2. Relative contribution of amino acid species in sieve tube sapand leaf extracts of Tanacetum. Mean ± SD, significance ***P < 0.001,**P < 0.01, *P < 0.05, t < 0.1. n sieve tube sap = 192, n leaf = 155.

Sieve tube sap LeafRatio (sieve tube

sap/leaf)

ala 0.0272 ± 0.0180 0.0586 ± 0.0315 0.464***

arg 0.125 ± 0.0724 0.0532 ± 0.0635 2.350***

asn 0.0743 ± 0.0586 0.0393 ± 0.0589 1.891***

asp 0.192 ± 0.146 0.130 ± 0.0631 1.477***

gaba 0.000686 ± 0.00292 0.0354 ± 0.0395 0.019***

gln 0.0749 ± 0.0641 0.103 ± 0.0652 0.727***

glu 0.174 ± 0.0537 0.216 ± 0.0882 0.806***

gly 0.0193 ± 0.0138 0.0800 ± 0.0599 0.241***

ile 0.0126 ± 0.00966 0.0162 ± 0.0113 0.778***

leu 0.00808 ± 0.00668 0.0144 ± 0.0118 0.561***

lys 0.0451 ± 0.0279 0.00940 ± 0.00896 4.798***

met/trp 0.00464 ± 0.0044 0.0164 ± 0.0272 0.283***

phe 0.0117 ± 0.0088 0.0172 ± 0.0101 0.680***

ser 0.165 ± 0.0902 0.113 ± 0.0437 1.460***

thr 0.0390 ± 0.0207 0.0436 ± 0.0202 0.894*

tyr 0.00878 ± 0.00722 0.00861 ± 0.0133 1.020val 0.0151 ± 0.00985 0.0209 ± 0.0124 0.722***

his, tyr and val were slightly accumulated (1.4–3-fold),the other amino acids were either at equal concentrationas in the leaves or even less, and gaba, gly and met/trpwere almost excluded from phloem sap (Table 3). Asimilar picture was seen in the relative distribution ofamino acids in phloem sap and leaves (Table 4), arg, glnand his were preferably taken up into the phloem, lys,ile and val were slightly preferred, the other neutral andthe acidic amino acids were hardly loaded (Table 4).

The cluster analysis grouped asn and arg close togetherwith met, thr and the branched-chain amino acids(Fig. 2A, Table S3), lys, ala and phe formed anothergroup. These amino acids were together with gabanegatively correlated to all other amino acids. Gly, aspand glu formed a group, whereas gln, tyr and ser wereapart from any other amino acid (Fig. 2A). The aminoacids in Ricinus leaves clustered differently, gln andarg grouped together and were negatively correlated tothe other amino acids, met/trp, tyr, phe and gly formeda group, thr, val, ile and leu another group (Fig. 2B,Table S4).

Discussion

The sieve tube sap from Tanacetum, which was obtainedafter stylectomy of aphids, showed a more than 10-foldaccumulation of some amino acids compared to leaves,although the real accumulation factor in Tanacetumsieve tube sap may be lower by 1.5 because of someevaporation during collection (Kallarackal et al. 2012).Evaporation is no topic for collection of bleeding sap

asp

glu

asn

gly

gln

ala

thr

leu tyr

phe

val

ile ser

met

/trp

gaba arg

lys1.0

0.5

0

–0.5

Cor

rela

tion

A Sieve tube sap

asp

glu

ser

gly

tyr

met

/trp ile val

leu

gln

thr

phe

ala

lys

gaba as

n

arg1.0

0.5

0

–0.5

Cor

rela

tion

B Leaf

Tanacetum vulgare

Fig. 1. Group average graph of the Pearson Correlation Resemblanceof amino acids in the sieve tube sap (A) and the leaf (B) of Tanacetumvulgare. The amino acids in each sample were standardized, i.e. theirrelative contribution within the total content of amino acids in the samplewas calculated. Then the software PRIMER6 was applied which created aso-called lower triangular resemblance matrix (resemblance measure:Pearson Correlation). From the resemblance matrix a hierarchical clusteranalysis was produced and a group average graph was created; n (sievetube sap) = 155, n (leaf) = 192.

from Ricinus, because of the large volumes of exudate.The leaf extracts cannot be further defined, but theamino acid concentration in leaf cell water is probablyclose to that of the cytosol (Riens et al. 1991, Winteret al. 1992). Since the concentration in the apoplastis only 1/10 or 1/100 of that in cytosol (Lohaus et al.(1995), probably all amino acids except γ -amino butyricacid were transported uphill from the apoplast into thephloem. The relative distribution of amino acid species,which is independent of evaporation effects, showed astatistically significant positive selection of some aminoacids into the phloem as well as a negative selectionof some other amino acids. No decision is possible,whether the specificity lied in the release of amino acidsinto the apoplast or in the uptake of amino acids from theapoplast into the companion cell-sieve tube complex.

The covariation analysis revealed which amino acidsformed a group by increasing or decreasing together.

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Table 3. Concentration of amino acids in sieve tube sap and leaf cellwater of Ricinus. Mean ± SD, significance ***P < 0.001, **P < 0.01,*P < 0.05, t < 0.1, n sieve tube sap and n leaf = 25 each.

Sieve tubesap (mM)

Leaf (μmol ml−1

leaf water)Ratio (sieve

tube sap/leaf)

ala 2.50 ± 2.38 11.27 ± 7.22 0.22***

arg 1.03 ± 0.60 0.61 ± 0.59 1.69**

asn 0.90 ± 0.62 2.07 ± 1.40 0.43**

asp 10.05 ± 16.94 22.06 ± 13.34 0.46*

gaba 0.95 ± 0.80 30.18 ± 24.01 0.03***

gln 73.90 ± 48.89 38.78 ± 44.10 1.91**

glu 15.42 ± 31.25 46.15 ± 22.58 0.33**

gly 0.63 ± 1.32 4.83 ± 3.71 0.13***

his 1.56 ± 0.53 0.51 ± 0.24 3.08***

ile 4.80 ± 3.61 4.80 ± 2.19 1.00leu 2.76 ± 1.44 4.97 ± 2.39 0.56**

lys 1.56 ± 0.64 2.82 ± 2.58 0.55t

met/trp 0.66 ± 0.57 12.48 ± 8.23 0.05***

phe 2.14 ± 2.75 5.55 ± 3.43 0.39**

pro 0.99 ± 0.27 3.11 ± 0.74 0.32***

ser 6.17 ± 7.21 10.52 ± 6.96 0.59thr 3.99 ± 2.97 7.34 ± 3.71 0.54**

tyr 0.23 ± 0.28 0.115 ± 0.207 2.00val 6.90 ± 5.87 4.78 ± 1.70 1.44*

Table 4. Relative contribution of amino acid species in the sieve tubesap and the leaves of Ricinus. Mean ± SD, significance ***P < 0.001,**P < 0.01, *P < 0.05, t < 0.1. n sieve tube sap = 39, n leaf = 29.

Sieve tube sap LeafRatio (sieve

tube sap/leaf)

ala 0.0234 ± 0.0233 0.0577 ± 0.0332 0.406***

arg 0.00817 ± 0.00455 0.00344 ± 0.00217 2.375*

asn 0.00626 ± 0.0029 0.0106 ± 0.00605 0.591***

asp 0.0550 ± 0.0341 0.0985 ± 0.0441 0.558***

gaba 0.00686 ± 0.00324 0.120 ± 0.0746 0.057*

gln 0.583 ± 0.117 0.190 ± 0.184 3.068***

glu 0.0790 ± 0.0568 0.214 ± 0.0851 0.369***

gly 0.00399 ± 0.00358 0.0215 ± 0.0219 0.186***

his 0.0137 ± 0.00295 0.00346 ± 0.00125 3.960***

ile 0.0355 ± 0.0185 0.0199 ± 0.0172 1.784***

leu 0.0211 ± 0.0114 0.0172 ± 0.0151 1.227lys 0.0142 ± 0.00900 0.00872 ± 0.00718 1.628**

met/trp 0.00445 ± 0.00456 0.0456 ± 0.0292 0.098***

phe 0.0176 ± 0.0116 0.0285 ± 0.0276 0.618*

pro 0.00903 ± 0.00251 0.0200 ± 0.00725 0.452***

ser 0.0400 ± 0.0119 0.0566 ± 0.0237 0.707***

thr 0.0293 ± 0.00874 0.0321 ± 0.0122 0.913tyr 0.00334 ± 0.00247 0.00865 ± 0.0127 0.386val 0.0439 ± 0.023 0.0285 ± 0.0255 1.540*

It was not a copy of the covariation in the leaves,which thus gave a hint to the specificity of amino acidtransporters. An exception was the group of branched-chain amino acids, which grouped together in leavesand sieve tube sap alike, but since they also have similaraffinities to the so-far characterized neutral amino acid

lys

ala

phe

gaba as

n

arg

met

/trp

thr

val

ile leu

gln tyr

ser

gly

asp

glu1.0

0.5

0

–0.5

Cor

rela

tion

A Sieve tube sap

Ricinus communis

gln

arg

met

/trp

gly

tyr

phe

thr

val

ile leu

ser

gaba gl

u

ala

lys

asp

asn1.0

0.5

0

–0.5

Cor

rela

tion

B Leaf

Fig. 2. Group average graph of the Pearson Correlation Resemblanceof amino acids in the sieve tube sap (A) and the leaf (B) of Ricinuscommunis. The amino acids in each sample were standardized, i.e. theirrelative contribution within the total content of amino acids in the samplewas calculated. Then the software PRIMER6 was applied which created aso-called lower triangular resemblance matrix (resemblance measure:Pearson Correlation). From the resemblance matrix a hierarchical clusteranalysis was produced and a group average graph was created. n (sievetube sap) = 29, n (leaf) = 39.

transporters, their grouping together may be the result ofthe broad specificity of these transporters.

Tanacetum is an apoplastic phloem loader (group2b) similar to some Brassicaceae like Lepidium and,probably, Arabidopsis (Gamalei 1989). Their phloemloading has to proceed through transporters, whichmakes accumulation and exclusion of some aminoacid species conceivable. The 2a-type in Ricinusphloem shows intermediate cells with smooth wallsto the inner side (without wall ingrowths and withoutplasmodesmata) and is a mixed symplastic/apoplasticphloem loader (Gamalei 1989). The generally loweraccumulation ratio of amino acids in Ricinus sieve tubesap compared to Tanacetum could be explained by somesymplastic transfer into the sieve tube sap. However,there is a selection of some amino acids, especially theexclusion of γ -amino butyric acid, therefore phloem

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loading must be by transporters, too, and not by asymplastic leak from the leaf cytosol. The smallerplasmalemma cross-sectional area of the intermediatecells toward the minor veins could be reason for a lowerrate of amino acid delivery to the sieve tube apoplast,another reason could be that a part of amino acids wereused up along the path from the leaf to the cut apex.

Arginine and lysine were more than 10-fold accumu-lated in sieve tube sap of Tanacetum and they werepositively selected from the leaf into the phloem. Bothamino acids showed close covariation. Thus there mustbe a transport system in the Tanacetum phloem, whichstrongly prefers these basic amino acids. Best candi-dates for transport of basic amino acids are transporterslike NtAAT1 or AtCAT1 (Frommer et al. 1995), whichhad been found in veins of source leaves (reviewed inFischer et al. 1998). The specificity of AtCAT1, whichprefers basic amino acids, but also accepts glutamine,valine and glutamate, would fit to the slight accumu-lation of these neutral amino acids, too, however itdoes not fit to the antagonistic covariation of glutamate.The sieve tube sap of Ricinus also showed a specificaccumulation of arginine, histidine and, though less, oflysine. These basic amino acids were also positivelyselected into the sieve tube sap, however arginine andlysine did not group together (the covariation of histidinecould not be proven because of low sample numbers).Arginine grouped with glutamine, isoleucine and valine,which were also positively selected. Thus the speci-ficity would agree with RcAAP1, which accepts basicand some neutral amino acids and which was found inveins, regrettably not in mature plants (Bick et al. 1998).Thus the phloem loading of basic amino acids by anAtCAT1-like transporter would be the best candidate forRicinus, too.

The results for the large number of neutral amino acidswere difficult to interpret, because most of these aminoacids were only barely accumulated, not much selectedand many of them grouped together. This was in someway expected, because the so far characterized aminoacid transporters AtAAP2, AtAAP4 and RcAAP3 showeda broad and not very distinct substrate specificity, evenAtAAP5 and At CAT1 show loading of neutral aminoacids as ‘side activity’. The small neutral amino acidsglycine, alanine and serine were slightly accumulatedin sieve tube sap of Tanacetum, but not in sievetube sap from Ricinus. The sieve tube sap of Ricinusis very rich in glutamine, which is accumulated andpreferentially selected compared to many other neutralamino acids, therefore a transporter with relatively highaffinity to glutamine has to be assumed. Gamma-aminobutyric acid was virtually excluded from sieve tube sapof both, Tanacetum and Ricinus. Therefore there was

no indication that a transporter like AtProT1, which islocated in Arabidopsis source leaves (Fischer et al. 1998,Breitkreuz et al. 1999) or AtGAT1 (Meyer et al. 2006) isactive in phloem loading in these plants.

The acidic amino acids glutamate and aspartate weremajor amino acids in Tanacetum sieve tube sap, butthey were less selected for phloem loading than forexample serine. Both grouped closely together andbehaved antagonistically to all neutral amino acids.Such an antagonistic behavior is only understood if abroad range transporter for both, neutral and acidicamino acids, was loading the sieve tubes of Tanacetum,whereas the affinity for the acidic amino acids is low.As only the molecules with protonated distal carboxylgroup are accepted by the transporters (Wyse and Komor1984, Fischer et al. 2002), which is a low percentageat the apoplastic pH, the neutral amino acids in theapoplast could compete-out the loading of the acidicamino acids and only a high concentration of theacidic amino acids in the apoplast could compensatethe low affinity. Transporter candidates are broad rangetransporters like AtAAP4 and AtAAP5, which were foundin veins (Fischer et al. 1998). A basically similar picturewas valid for sieve tube sap of Ricinus. Here, too,aspartate and glutamate closely grouped together (alsowith serine), but not antagonistic to other amino acids.Both acidic amino acids were only weakly accumulated.RcAAP3 with a broad specificity to neutral amino acidsand a low affinity to acidic amino acids fits to theseresults (Neelam et al. 1999).

Molecular transporter studies such as cloning and het-erologous expression plus substrate characterization arelacking for Tanacetum and only few transporters hadbeen isolated from Ricinus (Neelam et al. 1999). How-ever, a transporter equipment as found in Arabidopsismay be assumed to be valid for other plants, too, andtheir substrate characteristics might be similar though notidentical. Showing the presence of a transporter type byPCR using conserved primers would not add any furtherinformation on their function for phloem loading unlesslocalization and substrate specificity is studied, too.

In conclusion, phloem loading is accumulative andselective in both types of minor vein architecture,whether mixed symplastic/apoplastic as in Ricinusor pure apoplastic as in Tanacetum. There wasstrong evidence for an AtCAT1-like transporter to beresponsible for phloem loading of basic amino acids, andthere was strong evidence that AtProT1 or AtGAT1-liketransporters are absent in phloem loading, since γ -aminobutyric acid was almost excluded from phloem sap.

Acknowledgements – The work was financially supportedby DFG Graduiertenkolleg 678 (Herbivore) to S. N. B. and

Physiol. Plant. 2014

H. N., by DFG SEB Hoch-CO2 to F. K., by Humboldt-Foundation to J. K. and by FCI to E. K.

References

Bick JA, Neelam A, Hall JL, Williams L (1998) Amino acidcarriers of Ricinus communis expressed during seedlingdevelopment: molecular cloning and expressionanalysis of two putative amino acid transporters,RcAAP1 and RcAAP2. Plant Mol Biol 36: 377–385

Brady SM, Orlando DA, Lee JY, Wang JY, Koch J, DinnenyJR, Mace D, Ohler U, Benfey PN (2007) Ahigh-resolution root spatiotemporal map revealsdominant expression patterns. Science 318: 801–806

Breitkreuz KE, Shelp BJ, Fischer WN, Schwacke R, RentschD (1999) Identification and characterization of GABA,proline and quaternary ammonium compoundtransporters from Arabidopsis thaliana. FEBS Lett 450:280–284

Dinant S, Bonnemain J-L, Girousse C, Kehr J (2010)Phloem sap intricacy and interplay with aphid feeding.C R Biol 333: 504–515

Fischer W-N, Andre B, Rentsch D, Krolkiewicz S, TegederM, Breitkreuz K, Frommer WB (1998) Amino acidtransport in plants. Trends Plant Sci 3: 188–195

Fischer W-N, Loo DDF, Koch W, Ludewig U, Boorer KJ,Tegeder M, Rentsch D, Wright EM, Frommer WB (2002)Low and high affinity amino acid H+-cotransporters forcellular import of neutral and charged amino acids.Plant J 29: 717–731

Frommer WB, Hummel S, Unseld M, Ninnemann O(1995) Seed and vascular expression of a high-affinitytransporter for cationic amino acids in Arabidopsis. ProcNatl Acad Sci USA 92: 12036–12040

Gamalei Y (1989) Structure and function of leaf minorveins in trees and herbs: a taxonomic review. Trees 3:96–110

Girousse C, Bonnemain J-L, Delrot S, Bournoville R (1991)Sugar and amino acid composition of phloem sap ofMedicago sativa: a comparative study of two collectingmethods. Plant Physiol Biochem 29: 41–48

Girousse C, Bournoville R, Bonnemain J-L (1996) Waterdeficit-induced changes in concentrations in proline andsome other amino acids in the phloem sap of alfalfa.Plant Physiol 111: 109–113

Kallarackal J, Milburn JA (1984) Specific mass transfer andsink-controlled phloem translocation in castor bean.Aust J Plant Physiol 11: 483–490

Kallarackal J, Bauer SN, Nowak H, Hajirezaei M-R, KomorE (2012) Diurnal changes in assimilate concentrationsand fluxes in the phloem of castor bean (Ricinuscommunis) and tansy (Tanacetum vulgare). Planta 236:209–223

Lohaus G, Moellers C (2000) Phloem transport of aminoacids in two Brassica napus L. genotypes and one B.

carinata genotype in relation to their seed proteincontent. Planta 211: 833–840

Lohaus G, Winter H, Riens B, Heldt HW (1995) Furtherstudies of the phloem loading process in leaves of barleyand spinach: the comparison of metaboliteconcentrations in the apoplastic compartment withthose in the cytosolic compartment and in the sievetubes. Bot Acta 108: 270–275

Meyer A, Eskandari S, Grallath S, Rentsch D (2006)AtGAT1, a high affinity transporter forgamma-aminobutyric acid in Arabidopsis thaliana. J BiolChem 281: 7197–7204

Neelam A, Marvier AC, Hall JL, Williams LE (1999)Functional characterization and expression analysis ofthe amino acid permease RcAAP3 from castor bean.Plant Physiol 120: 1049–1056

Nowak H, Komor E (2010) How aphids decide what isgood for them: experiments to test aphid feedingbehaviour on Tanacetum vulgare (L.) using differentnitrogen regimes. Oecologia 163: 973–984

Pate JS (1980) Transport and partitioning of nitrogenoussolutes. Annu Rev Plant Physiol Plant Mol Biol 31:313–340

Riens B, Lohaus G, Heineke D, Heldt HW (1991) Aminoacid and sucrose content determined in the cytosolic,chloroplastic, and vacuolar compartments and in thephloem sap of spinach leaves. Plant Physiol 97:227–233

Tegeder M, Rentsch D (2010) Uptake and partitioning ofamino acids and peptides. Mol Plant 3: 1–15

Tegeder M, Ward JM (2012) Molecular evolution of plantAAP and LHT amino acid transporters. Front Plant Sci 3:1–11

Tilsner J, Kassner N, Struck C, Lohaus G (2005) Aminoacid contents and transport in oilseed rape (Brassicanapus L.) under different nitrogen conditions. Planta221: 328–338

Toufighi K, Brady SM, Austin R, Ly E, Provart NJ(2005) The botany array resource: e-northerns,expression angling, and promoter analyses. Plant J 43:153–163

Winter H, Lohaus G, Heldt H-W (1992) Phloem transportof amino acids in relation to their cytosolic levels inbarley leaves. Plant Physiol 99: 996–1004

Wyse RE, Komor E (1984) Mechanism of amino aciduptake by sugarcane suspension cells. Plant Physiol 76:865–870

Zurbriggen MD, Carrillo N, Tognetti VB, Melzer M,Peisker M, Hause B, Hajirezaei M-R (2009)Chloroplast-generated reactive oxygen speciesplay a major role in localized cell death during thenon-host interaction between tobacco andXanthomonas campestris pv. vesicatoria. Plant J 60:962–973

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1. Pearson Product Correlation of amino acidsin sieve tube sap from Tanacetum. The pair(s) ofvariables with positive correlation coefficients tend toincrease together, in the pairs with negative correlationcoefficients one variable tends to decrease while theother increases. For pairs with P-values greater than0.1, there is no significant relationship between the twovariables. tP < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001,number of samples 155 nd = could not be determined.

Table S2. Pearson Product Correlation of aminoacids in leaves from Tanacetum. The pair(s) ofvariables with positive correlation coefficients tend toincrease together, in the pairs with negative correlationcoefficients one variable tends to decrease while theother increases. For pairs with P-values greater than0.1, there is no significant relationship between the twovariables. tP < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001,number of samples 192, nd = could not be determined.

Table S3. Pearson Product Correlation of aminoacids in sieve tube sap from Ricinus. The pair(s) ofvariables with positive correlation coefficients tend toincrease together, in the pairs with negative correlationcoefficients one variable tends to decrease while theother increases. For pairs with P-values greater than0.1, there is no significant relationship between the twovariables. tP < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001,number of samples 29, nd = could not be determined.

Table S4. Pearson Product Correlation of amino acids inleaves from Ricinus. The pair(s) of variables with positivecorrelation coefficients tend to increase together, inthe pairs with negative correlation coefficients onevariable tends to decrease while the other increases.For pairs with P-values greater than 0.1, there isno significant relationship between the two variables.tP < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001, numberof samples 39, nd = could not be determined.

Edited by J. K. Schjørring

Physiol. Plant. 2014