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  BAÜ Fen Bil. Enst. Dergisi (2004).6.1 PHYSIOLOGICAL AND MOLECULAR ANALYSIS OF A VALINE RESISTANT MUTANT IN  ARABIDOPSIS THALIANA  Ekrem DÜNDAR Department of Biology , School of Arts and Sciences, Bal ı kesir University, 10100 Bal ı kesir / TURKEY ABSTRACT The passage of amino acids across membrane barriers in plant cells usually requires an amino acid transporter which links the transport to the proton motive force. In recent years many amino acid transporters have been characterized using various molecular and biochemical approaches. Another commonly used approach to identify genes, is to look for mutant phenotypes in T-DNA insertion mutagenized plants. A T-DNA tagged  Arabidopsis  seed population was screened with high concentrations of valine, and a putative valine transport mutant was isolated. Southern blotting displayed a single T-DNA insertion in the genome. Valine uptake deficiency of the mutant plant revealed by the whole seedling uptake assays using 14 C-labeled amino acids supports the hypothesis that the mutated gene is a valine transporter. Key Words:  Valine resistance, uptake deficiency, T-DNA mutagenesis. ÖZET Bitki hücrelerinde amino asitlerin membranlardan geçi ş i genellikle ta ş ı may ı  proton ta ş ı ma gücüne  ba ğ layan bir amino asit ta ş ı y ı c ı s ı  gerektirir. Son y ı llarda çe ş itli moleküler ve biyokimyasal yakla ş ı mlar kullan ı larak bir çok amino asit ta ş ı y ı c ı s ı  karakterize edilmi ş tir. Genleri tan ı mlamak için yayg ı nca kullan ı lan  bir ba ş ka yöntem ise T-DNA ile mutasyona u ğ rat ı lm ı ş  tohumlar  ı n aras ı nda mutant fenotipler aramakt ı r. Bu çal ı ş mada bir T-DNA mutant ı  tohum populasyonu yüksek valin ihtiva eden ortamda taramaya tabi tutularak  bir valin ta ş ı ma eksikli ğ i gösteren mutant bitki izole edildi. Southern Blot metoduyla yap ı lan analiz, mutant  bitkinin genomuna sadece bir T-DNA molekülünün eklendi ğ ini gösterdi. Radyoaktif karbonla ( 14 C) i ş aretlenmi ş  amino asitlerle yap ı lan ‘tüm fide ta ş ı ma’ deneyinin, mutant bitkinin dokular  ı na valin al ı m ı n ı n azalm ı ş  oldu ğ unu göstermesi, mutasyona u ğ ram ı ş  olan genin bir valin ta ş ı y ı c ı s ı  oldu ğ u hipotezini güçlendirmektedir. Anahtar Kelimeler:  Valine dayan ı kl ı l ı k, ta ş ı ma eksikli ğ i, T-DNA mutagenezi. 4  BAÜ Fen Bil. Enst. Dergisi (2004).6.1 1. INTRODUCTION 1.1 Valine Resistance A putative amino acid transport mutant was previously identified (1, 2). Unlike wild type seedlings that stop growing when germinated on media containing valine, this mutant  plant is able to survive. A unique feature about valine is that its biosynthesis is linked to that of leucine, and isoleucine. The pathways that synthesize valine, leucine and isoleucine all pass through the enzyme called acetolactate synthase or acetohydroxy acid synthase (AHAS). This enzyme is regulated by feedback inhibition by excess amounts of valine and leucine (3,4,5). Therefore, when either of these amino acids is in excess, AHAS stops the biosynthesis of all three branched amino acids (Figure 1), and the plant will die due to the starvation for the other amino acids (3). Based on this effect, valine resistant plants were screened in a T-DNA mutagenized population of  Arabidopsis . In this screen, seeds were germinated on 1 mM valine, and resistant plants were selected based on their ability to grow.  pyruvateacetolactate2-oxoisovalerate leucinevaline (-) (-) threonine(-) isoleucineAHAS 2 - acetohydroxybutyrate (-) AHAS   Figure 1. Regulation of AHAS through feedback inhibition of valine and leucine. Both valine and leucine alone can stop the biosynthesis of both amino acids, when in excess amounts. They bind to AHAS and block the first step in both leucine-valine pathway and in isoleucine pathway (adapted from Dey and Harborne, 1997). 5  BAÜ Fen Bil. Enst. Dergisi (2004).6.1 Resistance to high concentrations of valine could be due to a mutation in AHAS that results in the loss of feedback inhibition of the enzyme. As an alternative explanation a mutated transporter could also account for the resistant phenotype. The mutant transporter could be at the plasma membrane (blocks uptake) or it could be located in the plastid membrane blocking uptake to the stroma where AHAS is located (6, 7). It must be pointed out, however, that the mutated gene does not necessarily have to be a transporter but it could be a common regulatory gene for most (if not all) the amino acid transporters. Azaserine, a toxic amino acid analog, has been reported to be transferred by a leucine favoring amino acid transporter in animal cells (8). Since azaserine is a purine synthesis inhibitor (9), it is lethal to plants if taken up. In earlier work (1) the valine resistant mutant was also resistant to azaserine. This observation favors the ‘membrane transport mutant’ hypothesis over ‘a change in the target enzyme’. It is important to note that, many amino acid transporters with broad substrate specificity have been described in plants. Thus, it is now surprising this screen worked. This may suggest a key function and / or location of the  putative transporter identified in this screen. An explanation on the role of this putative amino acid transporter in nitrogen assimilate partitioning, and its unique role that allowed us to identify it using a mutant screen against high concentration of valine and against toxic amino acid analogues, would generate many useful insights toward understanding the physiology of plants. Taking the distinct role of amino acid transport in the plant’s life into account, this research would  provide a significant step forward in our understanding of amino acid transport as well as nitrogen assimilate partitioning in plants. 1.2 T-DNA Mutagenesis in  Arabidopsis Transferred DNA (T-DNA) is a part of the Ti plasmid of  Agrobacterium tumefaciens  that is transferred into the host plant genome during the infection by the  bacteria. The purpose of this transfer, as far as the bacterium is concerned, is to program the host genome so as to produce nutrients and structure (i.e. crown gall) that are essential for the bacteria. The bacterium does this programming by means of the genes encoded in the T-DNA that is transferred to the host genome (4, 10). Once transferred into the plant cell, the T-DNA part of the plasmid integrates itself into the nuclear genome through a special machinery that involves the participation of various proteins (11,12,13,14). Feldmann and Marks (15) generated a T-DNA tagged  Arabidopsis  seed library for mutant screening and made it available for distribution (16) through the Ohio State University  Arabidopsis  Biological Resource Center (ABRC), and through Nottingham  Arabidopsis  Stock Center (NASC) Department of Life Sciences. Since  Arabidopsis  introns are small and because there is very little intergenic material (17) in its genome, with a large enough population of T-DNA transformed lines, it is possible to saturate the genome with mutations in practically every gene. A study (18) reporting the random distribution of T-DNA integrations into  Arabidopsis  genome supports this hypothesis. More recent work (19) has indeed reported the near saturation of the gene space. The transport mutant 2607 was isolated (1) by screening the Feldmann tagged seed library for resistance against 1 mM valine. The T-DNA construct in Feldmann lines (15) and the restriction map of endonucleases used for Southern analysis, are shown in Figure 2. 6  BAÜ Fen Bil. Enst. Dergisi (2004).6.1 P r o b e BstZ17I  NdeI 5kb 8kb 4.5kb LBpBR322pBR322Tn9031’NPTRB BstZ17I  NdeI P r o b e Figure 2. The structure and the restriction map of the T-DNA construct used to generate 2607. BstZ17I and NdeI (New England Biolabs) cut T-DNA about 50 nucleotides apart from one another and hence they practically generate same size fragments. When double digestion products are probed as indicated, there will be 3 bands for a single insertion, 5 bands for a double insertion, 7 bands for 3 insertions, and so on. This approach can only detect the number of insertion sites which would very helpful to relate the phenotype to the insertion, but not the number T-DNA cassettes inserted side by side such as head to head or head to tail. The probe is a 3.5 kb fragment of pBR322.   2. MATERIALS AND METHODS 2.1  Arabidopsis  Growth Conditions  Arabidopsis thaliana  ecotype Wassilewskija (WS), and the T-DNA tagged  Arabidopsis  seed library (15, 20) was acquired from the  Arabidopsis  Biological Resource Center (ABRC) at The Ohio State University.  Arabidopsis  seeds were sterilized and grown  based on Sundaresan et al.(21). The seeds were soaked in 95% alcohol for 10 min, 20% Clorox 0.1% tween 20 for 5 min, washed with sterile water for 2 min twice and added 0.1% agar (top agar). The sterilized seeds were stratified 1-3 days and then transferred into plates that were made with 4.5 g•L -1  Murashige and Skoog (MS) salts (Invitrogen Corp., Carlsbad, CA, USA), 1% sucrose (Sigma-Aldrich, St. Louis, MO, USA), and 4.5 g•L -1  Agargel (Sigma-Aldrich, St. Louis, MO, USA) or 6 g•L -1  Bacto™ Agar (BD Biosciences, Boston, MA, USA). 1 mL from a 50 mg•mL -1  sterile kanamycin stock solution (50 µg•mL -1  final concentration), and valine to desired final concentration were added after media were cooled down to 55  o C, when needed. Plates with stratified seeds were grown in a controlled growth chamber that was set to 21  o C and 10 h 150 µ  E   m -2 •s -1  light cycle. Seedlings were transferred into well-watered Sunshine Mix soil (Wetsel Seed Co., Harrisonburg, VA, USA) when needed, and they were grown under controlled growth chambers that were set to 21  o C and 8 h 200 µ  E  m -2 •s -1  light cycle. 7  BAÜ Fen Bil. Enst. Dergisi (2004).6.1 2.2 Polymerase Chain Reactions and Sequencing Tail-PCR was performed as described (22) in a DNA-Engine PTC-200 (MJ Research, Inc., Watertown, MA). The PCR protocols and nested primers were as described  by Krysan et al.(23), and the arbitrary degenerate (AD) primers were as described by Liu and Whittier (22). The nested primers used in the first amplification were L1 (5’-GAT GCA CTC GAA ATC AGC CAA TTT TAG AC-3’) and R1 (5’-TCC TTC AAT CGT TGC GGT TCT GTC AGT TC-3’); and in the second amplification were L2 (5’-GGA TGT GAA TTC AGT ACA TTA AAA ACG TC-3’) and R2 (5’-GTC AGT TCC AAA CGT AAA ACG GCT TGT CC-3’). The AD primers used were AD1 (5’-NTC GA(G/C)T(A/T)T(G/C)G(A/T)GTT-3’), AD2 (5’-NGT CGA (G/C)(A/T)GANA(A/T)GAA-3’), AD3 (5’-(A/T)GTG NAG(A/T)ANCANAGA-3’). Two additional AD primers AD2a (5 '- STT GNT AST NCT NTG C-3') and AD5 (5’-WCA GNT GWT NGT NCT G-3’) (24) were also utilized when needed.  Nested primers for pTiC58 were pTiL1 (5’ACC TTC ACA TCC AGC ACA AGC ATA TCA-3’), pTiL2 (5’-ATC TTT GCG CAG CTC ATT CTT GAC CAA T-3’), pTiL3 (5’-TAG TTT TAT TGA TCA GCG GTT CCG CAA-3’), pTiR1 (5’-ACG AAA ATA TCC GAA CGC AGC AAG ATA T-3’), pTiR2 (5’-TTG AAG GCC AAA GCC TGG AAC TCA CTT T-3’), and pTiR3 (5’-TGT TTT TCG GAT GCC CGT TGA CGT ATT T-3’). Primers were designed using Primer3 software (25) on the web ( primer3_www.cgi), and ordered from Integrated DNA Technologies (Coralville, IA, USA). Inverse polymerase chain reaction (I-PCR) and the primers were as reported (26). All standard polymerase chain reactions were done using standard molecular biology  protocols (27) in a DNA-Engine PTC-200 (MJ Research, Inc., Watertown, MA). For sequencing the TAIL-PCR products directly from the second or third amplification, the brightest bands were gel extracted using a gel extraction kit (Qiagen Inc., Valencia, CA, USA), and sequenced by the Keck Center at the University of Illinois at Urbana-Champaign. 2.3 Southern Blotting Plant genomic DNA was isolated as described by Dellaporta et al. (28). Genomic DNA was digested with selected restriction enzymes, and separated on a 0.8% agarose gel in TAE buffer. After running approximately 3 h at 80 volts (constant), the gel was depurinated in 0.25 M HCl, denatured in 0.5 M NaOH / 1.5 M NaCl, and neutralized in 1 M Tris / 1.5 M NaCl (pH 8). Digestion products were transferred onto a Hybond N +  membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in 10 X SSC (standard saline citrate, pH 7) for 16-24 h, cross linked to the membrane with a UV cross-linker (Stratagene, La Jolla, CA, USA). α - 32 P-dCTP or was purchased from Perkin Elmer (Boston, MA, USA). 32 P-labeled probes were synthesized using a Mega Prime DNA labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Probes were cleaned up with Bio-Spin columns (Bio-Rad, Hercules, CA, USA), and probe specific activity was measured using a liquid scintillation analyzer (Packard Bioscience Company, Downers Grove, IL, USA). Pre-hybridization, hybridization and washing the membranes were done based on 8
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