Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds

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Research article

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds Q3

Galina V. Novikova a, Colette Tournaire-Roux b, Irina A. Sinkevich a, Snejana V. Lityagina a, Christophe Maurel b, Obroucheva Natalie a, * a b

Institute of Plant Physiology of Russian Academy of Sciences, Botanicheskaya str. 35, Moscow 127276, Russia INRA Montpellier SupAgro, F-34060 Montpellier, Cedex 2, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 January 2014 Accepted 24 May 2014 Available online xxx

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. © 2014 Published by Elsevier Masson SAS.

Keywords: Cell elongation Gene expression Seed germination Tonoplast aquaporins Vacuole biogenesis Vicia faba minor Water transport

1. Introduction Quiescent seeds are characterized by an absence of hormonecontrolled dormancy and their germination can simply be triggered by imbibition (Obroucheva, 2010, 2012; Weitbrecht et al., 2011; Bewley et al., 2013). Progressive water inflow results in tissue passing through successive thresholds of water content (WC), at which main metabolic processes are activated. A primary metabolic activation is completed at a WC of about 60% fresh weight (FW). Yet, a further increase in water content is necessary for growth initiation in embryonic axes. This process requires the accumulation of endogenous osmotica as well as cell vacuolation. These events, together with cell wall loosening, determine the initiation of embryonic axis extension and radicle protrusion. They

Abbreviations: FW, fresh weight; NIP, nodulin-like intrinsic protein; PIP, plasmalemma intrinsic protein; PSV, protein storage vacuole; TIP, tonoplast intrinsic protein; WC, water content. * Corresponding author. Tel.: þ7 499 231 83 30. E-mail addresses: [email protected], [email protected] (O. Natalie).

provide rapid and successful root contact with soil water, a prerequisite for advantageous germination. Whereas cell elongation is a fundamental process during early seed germination, the initiation of cell division varies between species and can occur simultaneously with elongation or later on (Obroucheva, 1999). Broad bean (Vicia faba L. var. minor) seeds are typical orthodox seeds, that is, they are capable of drying during maturation without any loss of viability. Their germination occurs first by pure cell elongation, whereas cell divisions start in the root meristem 15 h after radicle emergence (Obroucheva, 1999). These seeds are therefore a suitable model for studying the physiological processes underlying the initiation of germination. Previous work from our laboratory has provided an accurate description of growth initiation in germinating broad bean seeds (Obroucheva, 1999). These seeds exhibit hypogeal germination, during which cell elongation is initiated in the hypocotyl, to push the non-growing radicle tip and let it emerge through the seed coat. In terms of growth initiation, broad bean germination can be described as follows. In air-dry seeds, the embryonic axis consists of a radicle (2 mm) and a hypocotyl (2 mm), with a small plumula

http://dx.doi.org/10.1016/j.plaphy.2014.05.014 0981-9428/© 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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above it. In imbibing seeds, cell elongation is initiated at a water content of 72e73% WC, firstly in the upper hypocotyl, and spreads gradually to its base, thereby protruding the radicle tip. No cell division occurs in the hypocotyl. At radicle emergence, this organ is 3-mm-long, with its upper cells elongated to 100e110 mm. Elongation then begins in the emerged roots, in cells adjacent to the hypocotyl, whereas cell proliferation commences in the root meristem, when embryonic axes are 1 cm-long. In growing embryonic axes, cell division and elongation proceed in roots while the hypocotyl has already reached its final size of 9e10 mm, with fullyelongated 150 mm-long cells. Because of their role in protein storage and cell turgor, vacuoles potentially play a crucial role during seed maturation and germination. The biogenesis of vacuoles first as protein storage vacuoles and thereafter as large vegetative vacuoles has been described at € fte et al., 1992; Bolte et al., 2011; least partially in Arabidopsis (Ho Gattolin et al., 2011), pea (Robinson and Hinz, 1996) and pumpkin (Maeshima et al., 1994), but precise knowledge is still lacking in broad bean. Aquaporins, channel proteins that facilitate water transport across cell membranes, have emerged as important players in plant water relations (Maurel et al., 2008). In all plant species examined, aquaporins occur as numerous (>30) isoforms classified in at least 4 subfamilies. Members of the Plasma membrane Intrinsic Protein (PIP) and Tonoplast Intrinsic Protein (TIP) subfamilies represent the most abundant aquaporins in the plasma membrane and tonoplast, respectively. The PIP subfamily can be further divided in PIP1 and PIP2 sub-types. The Nodulin26-like Intrinsic Protein (NIP) and Small basic Intrinsic Protein subfamilies encode homologues involved in the transport of micronutrients or as yet unknown substrates. Because of the intrinsic link between water inflow and germination, the expression of aquaporins in germinating seeds was studied (Obroucheva, 2013), particularly in Arabidopsis (Willigen et al., 2006; Gattolin et al., 2011), rice (Liu et al., 2007, 2013; Li et al., 2008), Brassica napus (Gao et al., 1999), and their presence in embryonic axes of germinating broad bean seeds was preliminarily shown (Shijneva et al., 2007). Some of these studies have addressed the expression of the whole aquaporin family. In Arabidopsis, for instance, expression profiling using macro-array hybridization and immuno-blotting revealed that in dry seeds three TIPs (of which, two belong to the TIP3 subtype) were abundantly expressed, whereas no PIP expression could be detected (Willigen van der et al., 2006). Aquaporin expression was, however, dramatically altered during seed germination, the expression of the former TIPs vanished, whereas expression of seven PIPs and three TIP isoforms was progressively taking over. In rice, TIP3 homologues are also predominant in dry seeds and their expression is markedly reduced during germination (Li et al., 2008). Whereas genetic evidence is just emerging (Liu et al., 2007, 2011), the role of aquaporins during germination has been tentatively investigated using pharmacological inhibition. Mercurials, which are potent but unspecific and toxic aquaporin blockers, did not reveal any role for aquaporins during the early imbibition of pea seeds (Veselova and Veselovsky, 2006). In Arabidopsis as well, no inhibiting effect of mercury on seed water uptake was observed until expansion of embryonic tissues (Willigen van der et al., 2006). Whereas these data were obtained in intact seeds, studies in embryonic axes, in which the preparation and early initiation of growth take place, could provide a higher resolution. Therefore, we reasoned that broad bean seeds could represent a relevant system for studying germination and investigated the expression and function of plasma membrane and tonoplast aquaporins in embryonic axes during seed imbibition, radicle emergence and growth.

2. Materials and methods 2.1. Plant material Seeds of Vicia faba var. minor, cv. Streletskie, were provided by the Institute of Leguminous plants (Orel, Russia). Seeds being placed with their micropile down imbibed in distilled water in the dark at 27  C. Embryonic axes were excised from imbibing seeds, at radicle emergence, and during the post-germinative growth. Excised embryonic axes were fixed for light or electron microscopy or for protein and mRNA expression analyses. Cell length was measured in longitudinal sections along the third row of cortical cells. For electron microscopy, cross sections of embryonic axes were prepared according to standard procedure and examined under a Temscan-100 CX (Jeol, Japan) microscope. Cell vacuolation was estimated as the ratio of vacuole to cell area on electron micrographs. 2.2. Water content Water content (WC) was routinely measured by weighing prior to and after oven-drying for 1 h at 105  C and then at 80  C for 3 days and expressed as % FW. Water absorption by embryonic axes from killed seeds was measured after seed exposure to 105 C for 2 h, and to 80 C for a week. Rate of water uptake was calculated from the gain of water amount in the axis during imbibition. 2.3. Protein amount Protein amount was estimated using a BCA protein assay (Sigma, USA). 2.4. Isolation of membrane fractions Excised embryonic axes were homogenized in 300 mM sucrose, 10 mM EDTA, 5 mM potassium m-bisulfite, 5 mM DTT (dithiothreitol), 5 mM PMSF, 0.6% polyvinylpyrrolidone, 100 mM TriseHCl, pH 8.0. The homogenate was centrifuged at 10,000 g for 15 min; the supernatant was recovered and centrifuged at 100,000 g for 30 min. The resulting microsomal pellet was resuspended in 300 mM sucrose, 0.5 mM EDTA, 1 mM DTT, 10 mM MES-bis-TRIS propane, pH 7.2 and stored at 70  C. 2.5. Immunodetection Microsomal proteins were separated by SDS-PAGE followed by transfer onto Hybond C membranes (Amersham) as described by Towbin et al. (1979) with addition of 0.1% SDS. The blots were blocked in PBS containing 0.05% Tween 20 (PBST) and 5% nonfat dry milk, and incubated with appropriate primary antibodies at 4  C overnight. Since TIPs show a high level of homology in their Nterminal parts, antibodies were raised against selected peptides derived from the most variable C-terminal regions. Primary antibodies were raised in rabbits against a 18-amino acid (TNNMRPSGFHVSPGVGVG) peptide corresponding to residues 162e179 of Phaseolus vulgaris PvTIP3;1, and a 12-amino acid (NTTHEQLPTTDY) C-terminal peptide of AtTIP1;1. Because of the conservation of TIP sub-classes between plant species, it was assumed that these antibodies would cross-react with TIP homologues in V. faba (see supplemental Figure S1). Both TIP3;1 and TIP1;1 sequences were designed according to http://mbclserver. rutgers.edu/CPGN/AquuaporinWeb/Aquaporin.group.html. Polypeptides were synthesized in the Institute of bioorganic chemistry (Moscow) and antibodies were home-produced. The membranes

Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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were probed with horse radish peroxidase-labelled anti-rabbit antibodies in PBS. Cross-reactivity was visualized with SuperSignal Kit or with peroxidase color reaction. 2.6. Isolation and cloning of aquaporin cDNAs For total RNA extraction, the phenol-chloroform method of Downing et al. (1992) was used followed by a clean-up with RNeasy Mini Kit (Qiagen). In a first set of PCR amplification experiments, we used a combination of two degenerate oligonucleotides(50 -GGIGGICA(C/T)ITIAA(C/T)CCIGCIGTNAC-30 and50 -GCIGGICC(A/G)AAI(C/ G)(A/C/T)IC(G/T)ICGIGG(A/G)TT-30 corresponding to the first and second Asn-Pro-Ala (NPA) motifs, respectively) and possibly reacting with PIP and TIP (Gerbeau et al., 1999). In a second set of PCR amplification experiments, the second oligonucleotide was substituted by another primer. (50 -NGCIGG(A/G)TTCATI(G/C)(A/T)I(G/C)CICCI(G/C/T)(A/T/C)(A/ G)AA3-0 ), which was designed to match residues that are specifically conserved in the same region of TIP sequences (Zardoya and Villalba, 2001). PCR amplification was performed with one cycle of 4 min at 94  C, 45 s at 50  C and 1 min at 72  C, followed by 30 cycles of 45 s at 94  C, 45 s at 50  C and 1 min at 72  C. PCR products were gel-purified using a Geneclean III Kit (MP Biomedical, LLC). They were cloned in a pBSKII þ vector and the sequence of 22 and 19 cloned fragments from the first and second sets of amplification, respectively, was analyzed. RACE-PCR (30 -End cDNA Amplification) was performed according to Frohman (1995) with some modifications. RNAs were treated by RQ1 RNAse-free DNAse (Promega; 5U for 5 ml RNA at 37  C for 1 h) and 1 mg was used for production of cDNAs using MMLV reverse transcriptase (Promega), under conditions described by the manufacturer except for oligodT primer that was replaced by QT primer(50 -CCAGTGAGCAGAGTGACGAGGAC TCGAGCTCAAGCTTTTTTTTTTTTTTT-30 , Frohman, 1988). Two rounds of PCR amplification were performed as follows. A first round was realized using the above produced cDNA in the presence of 0.1 mM dNTP, 0.8 mM of a primer specific for each identified gene family (GSP1; Table 1) in combination with 0.8 mM of a Q0 primer (50 CCAGTGAGCAGAGTGACG-30 ) and 2.5 U Go-Taq polymerase (Promega). The PCR program included one cycle at 94  C, 5 min; 50  C, 5 min; 72  C, 10 min; 30 cycles at 94  C, 40 s; 50  C, 1 min; 72  C, 90 s and one cycle at 94  C, 40 s; 50  C, 1 min; 72  C, 15 min. PCR products were purified (GeneClean) and a second round of amplification was performed under the same conditions in presence of pairs of GSP2 (Table 1) and Q1 (50 -

Table 1 Gene specific primers (GSP) used for RACE amplification. Name

Sequence

TIP GSP1 TIP GSP2 PIP1 GSP1 PIP1 GSP2 PIP2 GSP1 PIP2 GSP2 TIP1;1 GSP1 TIP1;1 GSP2 TIP3;1 GSP1 TIP3;1 GSP2 TIP2;1 GSP1 TIP2;1 GSP2 TIP3;2 GSP1 TIP3;2 GSP2 NIP2 GSP1 NIP2 GSP2 new TIP2 GSP1 new TIP 2 GSP2

50 -GGTTTACACTGTCTATGCCAG-30 50 -AGCAGCTGACCCCAAAAAGG-30 50 -CCAGCCCAAGCAATACCAGG-30 50 -GCTCTAGGAGGAGGAGCTA-30 50 -GGTGCAATTTGTGGTGCTG-30 50 -GGACTAGCTAAGGGGTTCC-30 50 -CAGCATTCGGTCTATCCGC-30 50 -CAGGAGTAGGAGTGGGTCC-30 50 -CCAACAGGCCTTCAATCTGC-30 50 -CGAAAAATGTTGGTGCGGGA-30 50 -AAGAGTGTTCCAACCCATGG-30 50 -GAGTTGCTGCTGGATAAACC-30 50 -ACCAGCAGGGTTTCATGTAG-30 50 -GAGTGGGTCTAGGCGAAGGA-30 50 -CCATTGCTCACGCATCAACCA-30 50 -CCAAAAGATTTCCACCCAAAG-30 50 -CGGCTCAATAGTCGCATC-30 50 -CTCCTCAACTATGTCACCGCT-30

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GAGGACTCGAGCTCAAGC-30 ) primers, that overlap GST1 and Q0 primers. PCR products were purified, cloned in a pBSKII þ vector, and sequence was analyzed. For amino acid sequence comparisons, sequences from NPA to stop codon were aligned using MUSCLE V3.8.31 (Edgar, 2004). The maximum likelihood phylogenies were calculated using PhylML algorithm (Guindon et al., 2010) and the program Dendroscope (Huson et al., 2007, 2012) was used for building the tree.

66 67 68 69 70 71 72 73 74 75 2.7. Quantitative RT-PCR 76 77 Pairs of specific primers were designed in the 30 UTR of each of 78 the PIP and TIP isoforms identified (Table 2) using a PRIMER3 79 software (http://biotools.umassmed.edu/bioapps/primer3_www. 80 cgi). Real-time quantification of RNA was performed using a Light 81 Cycler II (Roche) essentially as described (Postaire et al., 2010). 82 Amplification was performed under the following conditions: 83 15 min at 95  C, followed by 40 cycles with 5 s at 95  C, 8 s at 84 62  C, 10 s at 72  C, and temperature transitions of 20  C/s. Cycle 85 threshold (Ct) values were determined by the fit point method 86 from the exponential phase of each amplification. Relative 87 quantification was determined using the Delta Ct method with 88 PCR efficiency correction and calibration with respect to dry 89 seeds. A mean Ct value was calculated from three independent 90 biological experiments, each with two PCR replicates. For 91 normalization, two reference genes (VfEF1, VfCYP2, AB012947) 92 were chosen on the basis of their expression stability in Vicia 93 faba seeds (Gutierrez et al., 2010) and two others (VfPP2a, 94 AB039917; Vf actin, AY338230) were selected on the basis of 95 stable expression of orthologs in seeds of soybean (Kulcheski et al., 2010) and Arabidopsis thaliana (Czechovski et al., 2005), Q1 96 97 respectively. The two more stable genes (CYP2 and Ef1) were 98 selected using a geNORM v3.4 software (Vandesompele et al., 99 2002), and a coefficient of variation was derived for data 100 normalization. 101 102 103 Table 2 104 Gene specific primers used for Q-RT-PCR. 105 Name Sequence 106 0 0 pip2for 5 -TCCCTGTTGAAGGTTTTGTCC-3 107 pip2rev 50 -ACAGGTTTGAGGACCCCATT-30 108 VF-TIP1;1-3b4 50 -CT GATTACTAGATGGGAAATGG-30 109 VF-TIP1;1-3b4rev 50 -CTCAAAAGAAAGATCCCTCCAA-30 VF-5TIP2;2for 50 -TGAGGGCTCTTTGTTCCAACC-30 110 VF-5TIP2;2rev 50 -CCATTTTTGAAAGAAGAGGTTC-30 111 0 0 VF-TIP2;2-3for 5 -GTGTTGTTGTTGGTACTTTGAAG-3 112 0 0 VF-TIP2;2-3rev 5 -AAGCAGTGGATAGAGAAAGAAC-3 113 VF-TIP3;1-21for 50 -GTTGCTTCCTAGCTTATGAAATG-30 114 VF-TIP3;1-21rev 50 -TTGAAGTGATAAAGGCTCACAC-30 VF-TIP3;2-14for 50 -ATGTGTGTGTTATAAGGGCATG-30 115 VF-TIP3;2-14rev 50 -CCCTGAGACCTTCAAAAACAGT-30 116 0 0 VF-NIP2for 5 -TTATCAAGGAATGTGGGAAGC-3 117 0 0 VF-NIP2rev 5 -GATGTTTTCCACTCTAGATTGAC-3 a 118 PIP1AAYfor 50 -TTCAAGTCCAGATCCTGATTTG-30 a PIP1AAYrev 50 -ACACATGATCCAGATCTCTCAA-30 119 b PIP1AJfor 50 -CATCTACAACAAAGACCATTCC-30 120 b PIP1AJrev 50 -CTCGAGGTCGACGGTATCGAT-30 121 PP2A1for 50 -TTGTCTGCTGCTGCTGCTGTC-30 122 0 0 PP2A1rev 5 -TTGCCGCTTCCTCCTATCAA-3 123 ACTfor 50 -TTTAACTGAGCGTGGCTACACT-30 ACTrev 50 -GAGCTAGTCTTTGCAGTTTCC-30 124 0 0 CYP2for 5 -TGCCGATGTCACTCCCAGAA-3 125 CYP2rev 50 -CAGCGAACTTGGAACCGTAGA-30 126 EF1Afor 50 -GTGAAGCCCGGTATGCTTGT-30 127 EF1Arev 50 -CTTGAGATCCTTGACTGCAACAT-30 128 Notes. a 129 Specific for AAY667436. b 130 Specific for AJ289701.

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2.8. Water uptake and effects of mercury Weighed excised embryonic axes were immersed in water or in a 0.75 mM HgCl2 for 30 min, then blotted, and weighed again, to evaluate the amount of absorbed water. Control axes were incubated in water until the end of experiment (90 min) and weighed. Some embryonic axes, initially exposed to HgCl2, were then rinsed in water and transferred to a 10 mM DTT. DTT-treated embryonic axes were blotted and weighed twice, at 60 and 90 min. Other embryonic axes that remained in the HgCl2 were also weighed twice. The minimal FW of sample for each treatment exceeded 350 mg. The experiments with broad bean axes were repeated 13 times and data represent means ± SE. 3. Results The kinetics of water inflow into embryonic axes during seed germination is shown in Fig. 1A.

Fig. 1. Hydration, growth initiation and cell vacuolation in embryonic axes of imbibing broad bean seeds. Arrows indicate radicle emergence. A. Hydration curves. 1 e water content; 2 e rate of water uptake. B. Growth curves. 1 e cell elongation and 2 e vacuolation in upper hypocotyl cells of imbibing broad bean seeds as a function of water content and imbibition time.

The imbibition curve of excised embryonic axes (curve 1) can be subdivided into three typical parts. The first steep increase in hydration level, up to 60% WC, is mainly due to saturation of the matric component of water potential. This process can be mimicked by embryonic axes from killed seeds, which can imbibe to up to 58 ± 1% WC (data not shown). The second part of the curve, from 12 h to radicle emergence at 24 h, can be described as a slow tissue hydration (from 60 to 74% WC). During this phase, water uptake is driven by a progressive accumulation of endogenous osmotica due to axis reserves, in particular, glucose and potassium ions (Obroucheva, 1999). The third portion of the hydration curve (t > 24 h) describes the gradual increase in WC in growing embryonic axes after radicle emergence, i.e. after growth initiation. This period is characterized by sucrose import from the cotyledons, progressive accumulation of osmotica and formation of large central vacuole. Based on these data, embryonic axes were sampled from imbibing seeds at 12 and 24 h, and at a length of 1 and 2 cm (15 and 30 h after radicle emergence). The curve 2 (Fig. 1A) represents the rate of water uptake by an embryonic axis. It shows a slow water uptake up to radicle emergence and a drastic enhancement of water inflow, concomitant with post-germinative cell growth. Cell length and vacuolation were characterized in embryonic axes to get closer insights into the cellular events associated with germination (Fig. 1B). Cell length increased sharply within a few hours prior to the radicle emergence. In dicotyledonous plants such as broad bean, dry seeds contain protein storage vacuoles formed at maturation from vacuoles. Curve 2 shows the course of cell vacuolation. The enlargement of vacuoles due to the transformation of protein storage vacuoles to typical vacuoles clearly occurred prior to the initiation of cell elongation. The pattern of vacuole biogenesis was explored by electron microscopy in embryonic axes of imbibing and germinating seeds. In cells entering elongation, vacuoles can account more than half of cell area due to numerous fusions and digestion of captured cytoplasmic inclusions (Fig. 1B). More specifically, dense protein storage vacuoles (PSV) gradually lost their contents due to proteolysis (Fig. 2A). The population of PSV within the hypocotyl cell was rather heterogeneous and their transformation to vacuoles did not occur concurrently. Their contents looked more and more flake-like (Fig. 2B). When their lumen became transparent, the restoration of PSV to vacuole was over (Fig. 2C) and they subsequently enlarged by fusion (Fig. 2D, E). Numerous vacuoles preceded the formation of a central vacuole (Fig. 2F). This pattern of vacuole formation was initiated in the upper hypocotyl cells of imbibing embryonic axis about 10 h prior to radicle emergence (Fig. 1B). It spread down to the hypocotyl base and was complete when the overall elongation of hypocotyl was accomplished. A distinctly different pattern of vacuole biogenesis was observed in growing embryonic axes after radicle protrusion whereby vacuoles developed from small provacuoles emerging from ER lagoons (Herman et al., 1994). More specifically, radicle basal cells contained large vacuoles previously formed from sparse PSV, side by side with small provacuoles distributed within the cytoplasm. These small vacuoles were especially numerous in radicle meristematic cells (Fig. 2G). In root cells formed de novo by meristem and entering elongation, these newly formed vacuoles enlarged (Fig. 2F), showing a wide range of sizes, and eventually fused (Fig. 2H, J). The intense water inflow that occurs during germination, the marked cell elongation that precedes and accompanies radicle emergence, as well as the profound reorganization of cell vacuoles described above prompted us to investigate the aquaporin equipment of germinating seeds. Putative aquaporin sequences were amplified by RT-PCR using degenerated primers corresponding to two conserved Asn-Pro-Ala (NPA) motifs present in plant

Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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Fig. 2. Electron micrographs of hypocotyl (AeF) and root (GeJ) cells in the course of germination. Designations: m e mitochondrion; psv e protein storage vacuole; pv e provacuole; v e vacuole. Arrowheads indicate vacuole fusion; black arrows (G, J) show provacuole appearance.

aquaporin homologues. Sequence analysis of 41 independently cloned PCR fragments revealed for all of them homology to previously described aquaporin sequences, and therefore allowed their classification within the PIP, TIP or NIP subfamilies. No SIP homologue was identified. To get more precise sequence information, cDNA ends corresponding to the above described PCR fragments were amplified by 30 RACE. The deduced translated sequences, from the first NPA motif to the stop codon were aligned together with the corresponding region of close Arabidopsis homologues and of already described Vicia faba PIP1s [VfPIP1-a, (Sun et al., 2001); VfPIP1-b, (Cui et al., 2005)].

Nineteen of the 41 broad bean amplified fragments were found to encode VfPIP2;1, a PIP homologue of PIP2 sub-type. As shown in the dendrogram of Fig. 3, the closest Arabidopsis homologues are AtPIP2;7 and AtPIP2;8). No clone corresponding to PIP1s, and to the two previously described VfPIP1-a and VfPIP1-b in particular, was identified. Five clones corresponding to TIP homologues fell into the previously defined TIP1 (VfTIP1;1), TIP2 (VfTIP2;1, VfTIP2;2) and TIP3 (VfTIP3;1, VfTIP3;2) sub-types (Fig. 3). Finally, one clone corresponding to a NIP homologue (VfNIP1;1) was also identified. 30 RACE cDNA ends of the above described PCR fragments were used to design primers in 30 untranslated sequences that are known

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Fig. 3. Amino acid sequence relationship of aquaporin sequences either deduced from cDNA fragments amplified from broad bean seeds or from representative homologues of Arabidopsis. GenBank accession numbers of previously described proteins are as follows: AtPIP1.1 (At3g61430), AtPIP1;2 (At2g45960), AtPI1;3 (At1g01620), AtPIP1;4 (At4g00430), At PIP1;5 (At4g23400), AtPIP2.1(At3g53420), AtPIP2;3 (At2g37180), AtPIP2;4 (At5g60660), AtPIP2;7 (At4g35100), AtPIP2;8 (At2g16850), AtTIP1.1 (At2g36830), AtTIP2.1 (At3g16240), AtTIP2;2 (At4g17340), AtTIP2.3 (At5g47450), AtTIP3.1 (At1g73190), AtTIP3;2 (At1g17810), AtNIP1.2 (At4g18910), AtNIP2.1 (At2g34390), VfPIP1a (AF266760), VfPIP1b (AY667436). The newly amplified sequences of Vicia faba were deposited to the European Nucleotide Archive (http://www.ebi.ac.uk/ena) and the following accession numbers were assigned : VfTIP1;1 (LK020674),VfTIP2;1 (LK020671), VfTIP2;2 (LK020672), VfTIP3;1 (LK020669), VfTIP3;2 (LK020670),VfPIP2;1 (LK020673), VfNIP1;1 (LK020675).

to be low conserved among aquaporin genes (Table 2). The VfPIP1-a and VfPIP1-b cDNAs were also considered in this study. Although the whole complement of broad bean aquaporins remains unknown, these primers were assumed to be gene-specific and used to probe for gene expression levels in quantitative RT-PCR analyses. Fig. 4 shows the relative abundance of the PIP and TIP aquaporin transcripts in embryonic axes during dry seed imbibition (0-h and 12-h), germination (radicle emergence at 24 h) and subsequent early growth (1- and 2-cm long embryonic axes). In agreement with the repartition of previously amplified PCR fragments, no expression of PIP1 was detected, whereas a strong q-T-PCR signal was observed for VfPIP2;1. However, the expression of this and VfNIP1;1 transcripts was noticeably stable during the germination, suggesting that these aquaporins fulfill housekeeping functions. By contrast, expression of all five TIP homologues was much more dynamic. Their time-dependent transcript expression profiles were further characterized, together with protein abundance, to search

for possible correlation with the kinetic patterns of vacuole biogenesis during germination. The abundance of both VfTIP3;1 and VfTIP3;2 mRNAs in embryonic axes declined during seed imbibition to reach a ~1000-fold reduced residual value after radicle emergence (Fig. 4). Expression level of TIP3s in embryonic axes was also investigated by western-blotting (Fig. 5A). Antibodies against an Phaseolus vulgare homologue revealed a typical band at 26 kDa that was apparent at 12- and 16-h imbibition (lanes 1e2), low at radicle emergence (lane 3) and disappeared in 1 cm-long growing axes (lane 4). By contrast to TIP3s, the abundance of VfTIP1;1 mRNA in embryonic axes gradually rose during germination and seedling establishment, by about 10-fold between the dry seed and 2-cmlong axes (Fig. 4). TIP1 proteins were below immunodetection level in imbibing seeds (Fig. 5B), but highly abundant in rapidly growing (2 cm) axial organs. The gradual increase in VfTIP1;1 expression,

Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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Fig. 4. Aquaporin gene expression in embryonic axes of imbibing and germinating broad bean seeds. The transcript abundance of each of VfPIP and VfTIP genes identified was measured by real-time RT-PCR. The mean expression ratio is shown in embryonic axes of dry seeds (0 h), 12-h imbibed seeds, at radicle emergence (24 h), in growing embryonic axes of 1 cm (39 h) and 2 cm (54 h). Expression ratios were calculated using a normalization factor calculated from the two most stable reference genes (VfCYP, VfEF1-a) among four and using dry seeds as a calibrator. Means (±SE) are from three independent biological experiments, each with duplicate PCR reactions.

and the high gene product content in rapidly growing roots were correlated with the appearance of small vacuoles in root tip and their enlargement as the root cells gradually elongate (Fig. 2). The mRNA abundance of both VfTIP2;1 and VfTIP2;2 showed the most pronounced increase by up to 1000-fold during the course of germination, with a sharp rise for VfTIP2;1 just after radicle emergence (Fig. 4). This timing coincides with active cell elongation in axial organs as seed germination proceeds. In 1e2-cm axial organs, the elongation culminates in root cells, whereas the hypocotyl extension decelerates. Unfortunately, we had no antiTIP2 antibodies to monitor the abundance of the VfTIP2 gene products. The expression patterns of individual aquaporins in broad bean embryonic axes point to the specific roles during seed maturation and germination, but do not allow concluding about their contribution to germination-associated water transport. To address this,

Fig. 5. Occurrence of tonoplast aquaporins in embryonic axes of imbibing and germinating seeds. A. Immunoblots and Coomassie-stained gels. B. Immunoblots with variable protein loading per lane. 1e12-h seed imbibition; 2e16-h imbibition; 3 e radicle emergence; 4 e 1 cm-long growing embryonic axes. 5 e growing embryonic axes of 2 cm.

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we investigated the effects of an aquaporin inhibiting treatment on water uptake by emerging and growing embryonic axes. Mercury ions are known to oxidize and bind to Cys of most aquaporins, thereby blocking water transport. Mercury elimination and treatment with a reducing molecule can reverse these effects. We compared the rates of water inflow after incubation in water and in 0.75 mM HgCl2 with the subsequent transfer into a 10 mM DTT. In 6e7-mm-long axes (at radicle emergence) exposure to HgCl2 for up to 90 min did not significantly alter the rate of water uptake (Fig. 6A). By contrast, water uptake in 1-cm-long axes was significantly reduced (by about 30% at 90 min) by mercury and these effects were fully reversed when DTT was substituted after 30 min. Therefore, the contribution of water channel proteins to water uptake becomes predominant only after radicle emergence, i.e. when cells begin to actively elongate. These cells are characterized by intense water uptake as indicated in Fig. 1A (curve 2) and Fig. 6 (0.7 mg/90 min at radicle emergence, (Fig. 6A) vs. 4.3 mg/90 min at 1-cm stage (Fig. 6B). We note that this intense water uptake corresponds to the onset of expression of TIP2 aquaporins. 4. Discussion 4.1. Cell vacuolation in germinating seeds Here we provide a comprehensive description of structural and molecular events that occur during the course of broad been seed germination. A special focus was brought onto the overall kinetics of cell vacuolation. Two paths for vacuole biogenesis have been

Fig. 6. Water absorption by embryonic axes excised from broad bean seeds. Arrows indicate the transfer from 0.75 mM HgCl2 to 10 mM dithiothreitol (DTT). Upper curves show water absorption by control embryonic axes. A. Embryonic axes of 6e7 mm (radicle emergence).B. Embryonic axes of 1 cm.

Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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described in seeds, namely restoration from protein bodies and formation from enlarging endoplasmic reticulum (ER) cisternae (Marty, 1999). The first path is typical of dicotyledonous plants, dry seeds of which contain PSV formed at maturation from vacuoles. The transformation of PSV to typical vacuoles in broad bean seeds occurred as partly exemplified in a series of electron micrographs (Fig. 2 AeC). In imbibing embryonic axes, PSV had a rounded appearance with an average diameter of 2 mm, and exhibited an electron-dense and homogenous matrix in the interior. These organelles lack the dense central globoids containing phytin (Weber and Neumann, 1980) described in other legume species, and their contents represents uniformly dispersed reserve proteins, mainly globulins. In embryonic axes, proteolysis of reserve proteins was triggered when a range of 45e55% WC was reached (Obroucheva, 1999) and was likely carried out by stored proteinases (Muntz et al., 2001). Within the range of 55e65% WC, solubilization and early degradation of globulins, mainly legumins, occurs (Lichtenfeld et al., 1979), apparently due to endopeptidases delivered from cytoplasm (Muntz et al., 2001). At 65% WC, the average size of PSV rose to about 3 mm in diameter, their matrix becoming dispersed and lacelike. Their further transformation to vacuoles included fusion and enlargement events occurring at 68e71% WC (Fig. 1A). This pattern of cell vacuolation is characteristic of hypocotyls (Fig. 2 AeF), which are the first to elongate during the hypogeal germination of broad bean seeds. A distinct pattern of vacuole biogenesis was observed in radicle cells (Fig. 2 GeJ). Their extension occurred, first in basal cells, and subsequently in the cells formed by the meristem, when the radicle transforms into a root. In the basal cells, PSV are rare, and their transformation to vacuoles proceeds in parallel with de novo development of small provacuoles. In contrast, provacuoles are numerous in basal meristematic cells of the radicle (Fig. 2G) and they develop to normal vacuoles upon root cell elongation (Fig. 2H, J). A similar course of vacuole biogenesis was observed by Herman et al. (1994) in oat root cells. Thus, our data point to two parallel pathways of vacuole biogenesis in embryonic axes of germinating broad bean seeds. Vacuolation via PSV transformation is typical of the hypocotyl. This organ is initially used for storage and its growth is determinate, with a fixed number and final length of constituting cells. In contrast, the formation of provacuoles from the ER and their fusion and enlargement to vacuoles is typical of radicle/root cells, and more generally of indeterminate growth, as continues in the root after germination beginning. Nevertheless, independent of the patterns of vacuole biogenesis in two organs of embryonic axis, the analogous central vacuole develops in fast elongating cells of growing hypocotyl and root. Overall, we believe that this refined knowledge of vacuole dynamics in broad been seeds provides a useful frame to integrate the molecular and physiological processes associated to cell expansion and water transport. 4.2. Aquaporin expression By contrast to other plant species, restricted knowledge was initially available on aquaporins of Vicia faba with only two PIP1 sequences deposited in database. We were able to amplify cDNA fragments of seven novel isoforms including one PIP2, five TIPs and one NIP. The expression of these and the PIP1 isoforms was probed using q-RT-PCR and when possible with isoform-specific antibodies. In view of the high number of PCR-amplified cDNA fragments and of the strong q-RT-PCR signal, VfPIP2;1 represents one of most highly expressed aquaporins in the embryonic axis. The failure to immunodetect this isoform using an antibody against a C-terminus

of AtPIP2;1 (data not shown) is likely due to a local sequence divergence between the broad bean and Arabidopsis isoforms. Although the transcript abundance of VfPIP2;1 (and supposedly protein abundance) was fairly constant during seed germination, it cannot be excluded that the activity of this aquaporin was modulated by phosphorylation, as shown in other PIP2 homologues (Maurel et al., 2008). By contrast to VfPIP2;1, no transcript for PIP1s could be PCR-amplified suggesting that this subtype of aquaporins is low abundant in broad bean embryonic axes. Yet, this sub-type of aquaporins may be limiting in other species since altered expression of OsPIP1;1 and OsPIP1:3 in transgenic rice revealed a positive link between these two aquaporins and seed germination (Liu et al., 2007, 2013). In relation with the precise patterns of vacuolar biogenesis, we also found that broad bean seeds show well-defined kinetic expression profiles of several TIP isoforms. a-TIP (now called TIP3;1) and close homologues of the TIP3 subtype were first identified in the tonoplast of protein storage vacuoles in cotyledons of legumes and pumpkin (Johnson et al., 1989; Melroy and Herman, 1991; Herman and Larkins, 1999; Jauh et al., 1999), (Inoue et al., € fte et al., 1992; Willigen 1995), in intact seeds of Arabidopsis (Ho et al., 2006; Hunter et al., 2007; Bolte et al., 2011) and aleurone grains of rice (Takahashi et al., 2004; Li et al., 2008). These aquaporins start accumulating in protein storage vacuoles, after the bulk of reserve proteins has been stored (Johnson et al., 1989; Melroy and Herman, 1991). Although a transient localization of TIP3s in the plasma membrane of maturing Arabidopsis seeds was recently described (Gattolin et al., 2011), TIP3s are considered as typical markers of protein storage vacuole membranes (tonoplasts) (Hunter et al., 2007). In agreement with previous work, showing that TIP3s progressively disappear during germination (Takahashi et al., 2004; Willigen et al., 2006; Hunter et al., 2007; Li et al., 2008; Bolte et al., 2011; Gattolin et al., 2011), we observed that the abundance in broad bean embryonic axes of VfTIP3;1 and VfTIP3;2 transcripts showed a sharp decrease during seed imbibition to a residual level after radicle emergence (Figs. 4 and 5A). Consistent with this, a reduced abundance of TIP3 immunorelated proteins and their degradation in elongating embryonic axes accompanied the progressive transformation of protein storage vacuoles to large vacuoles. Although the precise function of TIP3s awaits elucidation, our data support the idea that these aquaporins are specifically related to water status maintenance in maturing seeds. Water displacement due to reserve protein deposition and concomitant remodeling of the vacuolar apparatus may require specific regulation of water transport across the tonoplast. We note that, in some recalcitrant seeds such as horse chestnut, protein storage vacuoles are absent, active vacuoles are preserved, and embryonic axes of mature seeds maintain vacuolar expression of TIP3s during imbibition, germination and growth (Obroucheva et al., 2012). Members of the TIP1 subtype were initially associated with cell growth (Ludevid et al., 1992). This was confirmed in numerous studies whereby TIP1s appeared as the most widespread TIPs in roots and shoots (Hunter et al., 2007; Gattolin et al., 2009). AtTIP1;1 (previously called g-TIP) is absent from Arabidopsis embryos (Willigen et al., 2006; Hunter et al., 2007; Bolte et al., 2011) but was recently found in the seed coat, within the chalaza region, where the conducting tissues terminate (Gattolin et al., 2011). Its expression in seedlings started at about two days after seed germination (Takahashi et al., 2004; Willigen et al., 2006; Hunter et al., 2007; Gattolin et al., 2011). Consistent with this, enhanced expression of VfTIP1;1 was detected in germinating broad bean seeds, first at the transcript level (Fig. 4). A marked enhancement of TIP1 protein content was subsequently observed, in rapidly growing embryonic axes of 2 cm (Fig. 5B).

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Although AtTIP2;1 was found to be expressed in the Arabidopsis seed coat, TIP2s seem to be virtually absent in the embryo (Gattolin et al., 2011). In Arabidopsis seedlings, TIP2 gene expression raises at two days after radicle emergence (Willigen van der et al., 2006), following the enhancement of AtTIP1;1 expression (Hunter et al., 2007). In rice seedlings, OsTIP2;2 expression increases on day 5 after germination (Li et al., 2008). According to Hunter et al. (2007), TIP2s would be expressed in mature conducting tissues of vegetative organs, i.e., after the completion of cell elongation. A somewhat earlier expression pattern was observed in broad bean seeds with a sharp rise in transcript accumulation that accompanied rapid cell elongation (Fig. 4). Thus, TIP2s appeared in the present study as the aquaporin isoforms, the expression of which was the most tightly linked to cell expansion. The overall results experimentally support the assumption that, during germination, changes in aquaporin composition in embryonic axes are closely related to changes in the pattern of vacuolar biogenesis. 4.3. Functioning of aquaporins in germinating seeds In growing organs, and in the absence of suitable genetically altered materials, pharmacological inhibition remains the only accessible technique for probing the functioning of aquaporins. Here, this approach consisted of short-term measurements of water absorption and treatments by HgCl2, with subsequent recovery from possible mercury inhibition by DTT (Barrowclough et al., 2000; Javot and Maurel, 2002). When applied to broad bean embryonic axes (Fig. 6A), no mercury inhibition was observed at radicle emergence. However, shortly after growth initiation (embryonic axes of 1 cm), a marked, reversible inhibition of water uptake by HgCl2 was observed (Fig. 6B). Similar results were obtained with embryonic axes from horse chestnut seeds (Obroucheva et al., 2012), namely no inhibition of water uptake at radicle emergence and a strong inhibition of water inflow to axial organs growing by elongation. The differential effects of HgCl2 on two growth events support the notion of an essential role for aquaporins in the fast cell expansion. Such distinction can be related to differences in the absolute rate of water absorption (Fig. 1A, curve 2). During imbibition up to radicle emergence, the rate of water inflow into embryonic axes was low, but increased by ten-fold, when cell elongation was promoted. Apparently, water uptake prior to and during radicle emergence occurs mostly by diffusion, without major contribution of water channels, whereas later on, due to active accumulation of endogenous osmotica, facilitated water influx through active water channels would enhance the enlargement of vacuoles and would support the elongation of cells. Whereas PIPs have been hypothesized as the main targets of mercury inhibition in plant tissues, our mRNA expression analyses indicate that VfPIP2;1 was highly abundant throughout the germination process and therefore was not specifically implicated in the fastest phases of cell expansion. This suggests that cell-tocell water transport was not specifically limiting during this process. Yet, it cannot be excluded that as yet uncharacterized PIP genes would encode mercury sensitive aquaporins specifically contributing to cell expansion. Nevertheless, our structural studies indicated that the fastest phases of cell expansion were accompanied by a profound reorganization of the vacuolar system. This finding and the strong expression of TIP1 and TIP2 aquaporins sensitive to HgCl2 (Daniels et al., 1996) indicate that they may be the primary targets of HgCl2 and the likely candidates for meeting the requirements of rapid growth in germinating seeds. More generally, our study emphasizes the central role of vacuolar dynamics during plant cell extension. In general, seed germination

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starts by slow elongation of embryonic axis cells followed by a rapid one. In terms of this paper, the initiation of cell elongation is characterized by a low rate of water inflow by diffusion, the presence of aquaporin water channels in closed state and slow vacuole biogenesis, the prerequisite of cell elongation beginning. During rapid cell elongation, the up-regulated expression of aquaporin tonoplast genes aimed at the increase in water channel number is coupled with accelerated water inflow through opened water channels made by TIP1 and TIP2 aquaporins. This results in active vacuole enlargement regardless of previous vacuole biogenesis pattern, thus providing fast cell extension and successful germination. Acknowledgments The work was supported by the Program of the Presidium of Russian Academy of Sciences “Cellular and Molecular Biology”, by Russian Federation of Basic Research [grant numbers 08-04-00416, 11-04-01139], by a CNRS/RAS cooperation agreement [grant num€l ber 21263 “Aquaporins and seed germination”]. We thank Michae M. Wudick for assistance in western blotting experiments, and cile Fizames for her help in phylogenetic analyses and sequence Ce submission. Contribution Cellular growth analysis, electron microscopic studies and experiments with mercury chloride were made by N. Obroucheva and S. Lityagina, isolation of microsomal fractions and western blotting were carried out by I. Senkevich, whereas the identification of aquaporin genes and analysis of gene expression were performed by C. Maurel, C. Tournaire-Roux and G. Novikova. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.05.014. Uncited references Obroucheva and Sinkevich, 2010. References Barrouclough, D., Peterson, C., Steudle, E., 2000. Radial hydraulic conductivity along developing onion roots. J. Exp. Bot. 51, 547e557. Bewley, J.D., Bradford, K.J., Hilhorst, H.W.M., Nonogaki, H., 2013. Seeds: Physiology of Development, Germination and Dormancy. Springer, New York. Bolte, S., Lanquar, V., Soler, M.-N., Beebo, A., Satiat-Jeunemaitre, B., Bouhidel, K., Thomine, S., 2011. Distinct lytic vacuolar compartments are imbedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds. Plant Cell Physiol. 52, 1142e1152. Cui, X.H., Hao, F.S., Chen, H., Cai, J.H., Chen, J., Wang, X.C., 2005. Isolation and expression of an aquaporin-like gene VfPIP1 in Vicia faba. Prog. Nat. Sci. 15, 496e501. Czechovski, T., Stitt, M., Altmann, T., Udvardi, M.K., Schreible, W.R., 2005. Genomewide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5e17. Daniels, M.J., Chaumont, F., Mirkov, T.E., Chrispeels, M.J., 1996. Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site. Plant Cell 8, 587e599. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792e1797. Frohman, M.A., 1995. Rapid amplification of cDNA ends. In: Diffenbach, C.W., Dveksler, G.S. (Eds.), PCR Primers, a Laboratory Manual. Cold Spring Harbor Laboratory Press, pp. 381e409. Gao, Y.P., Young, L., Bonham-Smith, P., Gusta, L.V., 1999. Characterization and expression of plasma and tonoplast membrane aquaporins in primed seeds of Brassica napus during germination under stress conditions. Plant Mol. Biol. 40, 635e644.

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Please cite this article in press as: Novikova, G.V., et al., Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.05.014

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