Patent Application: US-99876004-A

Abstract:
the present invention relates to a gene encoding a protein named as aria , which contains armadillo repeats and a btb / poz domain . the protein is a novel aba signaling component , which affects aba - regulated gene expression , seedling growth , aba sensitivity , and stress tolerance of plants . furthermore , the present invention provides a method of producing salt - tolerant plants comprising the introduction of an expression cassette containing the aria gene linked to a plant promoter to plants .

Description:
we carried out yeast two - hybrid screens to isolate abf2 - interacting proteins ( chien et al ., 1991 ; gyuris et al ., 1993 ). since abf2 has transcriptional activity ( choi et al ., 2000 ), bait constructs were prepared employing partial fragments of abf2 ( fig1 a ), to reduce the background activity . a cdna expression library representing rna from aba - treated arabidopsis seedlings ( choi et al ., 2000 ) was then used to transform a yeast strain containing each bait construct . we recovered five positive clones that interacted with the variable region ( amino acids 234 - 337 ) of abf2 . insert analysis of the clones showed that two of them ( group 1 ) encoded a transcription factor , which will be reported elsewhere . remaining three clones ( group 2 ) encoded an arm repeat protein ( see below ). the group 2 clones did not interact with nuclear lamin or with the corresponding regions of abf3 and abf4 ( fig1 b ), indicating that they specifically interacts with abf2 . the longest open reading frame ( orf ) of the group 2 clones encoded a protein containing 705 amino acid residues . the orf was missing the initiation codon . database search and subsequent isolation / sequencing of the full - length cdna ( seq id no : 1 ) revealed that the protein consists of 710 amino acid residues with a predicted molecular weight of 78 kd ( seq id no : 2 ) ( fig1 c ). the abf2 - interacting protein , designated as aria ( arm repeat protein interacting with abf2 ), has 9 copies of arm repeat in the n - terminal half , with arm 1 , 8 , and 9 being less - well conserved . additionally , it has a btb / poz domain in the c - terminal region . the gene encoding aria ( at5g19330 ) is composed of 19 exons and aria exhibits the highest sequence identity ( 59 %) to another arabidopsis arm repeat protein ( at5g13060 ) of unknown function . the interaction between aria and abf2 was confirmed by in vitro binding assay . recombinant proteins ( fig1 d , lanes 3 - 5 ) containing the entire aria coding region , the arm repeat region , or the btb domain as a fusion to the glutathione - s - transferase ( gst ) were prepared . their interaction with the full - length abf2 was then determined by gst pulldown assay , using in vitro translated abf2 labeled with 35 s . as shown in fig1 d , abf2 was retained by the gst - full - length aria fusion protein ( lane 7 ), whereas it was not retained by gst alone ( lane 6 ). thus , full - length aria interacted with abf2 . similarly , the fragments containing the arm repeat region or the btb domain also interacted with abf2 ( lanes 8 and 9 ). the stronger band intensity observed with the btb domain ( lane 9 ) suggested that abf2 bound the domain more strongly . expression patterns and subcellular localization of aria are similar to those of abf2 the aba - and stress - inducibility of aria expression was examined by rna gel blot analysis . like abf2 , whose expression is induced by aba and high salt ( choi et al ., 2000 ), aria transcript level was enhanced by aba and high salt treatments ( fig2 a ). to investigate the temporal and spatial expression patterns of aria in detail , histochemical β - glucuronidase ( gus ) staining of transgenic plants carrying an aria promoter - gus fusion construct was conducted . strong gus activity was detected in the radicles of germinating seedlings ( data not shown ) and in the roots of young seedlings ( fig2 b , a ). in older seedlings ( fig2 b , b ), leaves exhibited stronger gus activity than roots . in particular , the vascular tissues and the guard cells were stained strongly ( fig2 b , c ). in roots of older seedlings , gus activity was detected mainly in lateral roots rather than in the primary roots ( fig2 b , d ). the vascular region was more strongly stained than the epidermal tissues ( fig2 b , e , upper panel ), and very strong gus activity was observed in lateral root primordia and in the basal part of the lateral roots ( fig2 b , e , lower panel ). anthers , filaments , stigma , and the abscission zone of immature siliques exhibited strong gus activity among the reproductive organs ( fig2 b , f - h ). embryos were also stained strongly ( fig2 b , a , inset ). in summary , aria promoter activity was detected in embryos and most of the vegetative and reproductive organs . the temporal and spatial expression patterns of aria are very similar to those of abf2 . for instance , abf2 promoter is very active in most of the vegetative tissues , especially , in lateral roots , leaf veins , and guard cells . besides , abf2 is also strongly expressed in anthers , filaments and stigma among the floral organs . abf2 is a transcription factor and , as shown in fig2 c ( top panel ), is localized in the nucleus . we noticed that aria has a nuclear localization signal near its n - terminus ( fig1 c ), suggesting that it may be localized in the nucleus . to determine the intracellular localization of aria , transgenic plants harboring an aria - gfp ( green fluorescent protein ) fusion construct were generated , and the localization of the fusion protein was determined . fig2 c ( middle panel ) shows that gfp was localized in the nucleus , indicating that aria is nuclear - localized . gfp was also detected in the periphery of cells . it appears that aria is localized in the cell membrane as well . to investigate the in vivo function of aria , we generated and analyzed aria overexpression lines . transgenic arabidopsis plants expressing aria under the control of 35s promoter were generated ( see methods ), and , after preliminary analysis of seven t3 homozygous lines , aba / stress - related phenotypes of two representative lines were investigated in more detail . aria overexpression lines did not exhibit significant growth phenotypes under normal condition except slightly (˜ 1 hr ) delayed germination ( data not shown ). however , aria overexpression affected aba sensitivity during germination . aba dose - response analysis ( fig3 a ) showed that germination of 35s - aria transgenic seeds was more severely inhibited by aba than wild type seeds , especially at medium concentrations ( 1 and 2 μm ) of aba . thus , aria overexpression enhanced aba sensitivity during seed germination . in addition , germination of the transgenic seeds was more sensitive to mannitol , glucose , and nacl ( fig3 b ), indicating that aria overexpression resulted in hypersensitive response to high osmolarity . we also investigated the responses of 35s - aria seedlings to various abiotic stresses and found that they are less sensitive to high salt . for example , the survival rate of wild type plants at 100 mm nacl was 55 %, whereas those of 35s - aria plants were 81 % ( ar40 ) and 72 % ( ar32 ), respectively ( fig3 c ). at 125 mm nacl , 38 % ( ar40 ) or 36 % ( ar32 ) of the transgenic plants survived , whereas the wild type survival rate was 11 %. thus , aria overexpression lines were more tolerant to high salinity condition . to gain further insights into the in vivo function of aria , we analyzed the aria mutant phenotypes . a mutant , in which a t - dna is inserted in the promoter region of aria ( fig4 a ), was obtained from the arabidopsis stock center and , after the confirmation of t - dna insertion ( see methods ) and the abolishment of aria expression ( fig4 a ), various phenotypes were scored . germination assay ( fig4 b ) showed that the mutant seeds germinated more efficiently than wild type seeds under normal growth condition , although the degree of difference was not high . postgermination growth of the aria mutant was also more efficient ; i . e ., aria seedlings were larger than wild type plants , as shown in fig4 c . they developed normally , however , and the fully - grown mutant seedlings were of similar size to the wild type plants , indicating that the mutation affected the growth of young seedlings only . together , the observations demonstrate that aria is a negative regulator of seed germination and young seedling growth . the aria mutant also exhibited altered aba response . aba dose - response analysis of germination ( fig4 d ) revealed that the mutant seed germination was les sensitive to aba inhibition than wild type seeds at high concentrations of aba ( i . e ., 2 and 5 μm ), indicating that their germination was partially insensitive to aba . similarly , primary root elongation of aria plants was less sensitive to aba - inhibition than wild type plants at higher aba concentrations ( i . e ., 2 , 5 and 10 μm ) ( fig4 e ). glucose inhibits the shoot development ( i . e ., cotyledon greening , cotyledon expansion , and true leaf formation ) at high concentrations , and the inhibition process is dependent on aba ( jang et al ., 1997 ; leon and sheen , 2003 ). to see whether aria is involved in the process , we determined the glucose sensitivity of aria plants . fig4 f shows that cotyledon greening of wild type plants was gradually inhibited as glucose concentration in the medium increased . the aria mutant plants were also responsive to glucose in a similar manner , but the degree of inhibition was lower than that of the wild type plants . the differential response was not observed with mannitol , i . e ., it was not osmotic response ( data not shown ). the result demonstrates that aria is a necessary component for the glucose - inhibition of shoot development . to examine whether aria affects abf2 - regulated gene expression , we determined the expression levels of a number of abf2 - responsive genes in 35s - aria plants . coupled reverse transcription and polymerase chain reactions ( rt - pcr ) ( fig5 ) showed that the rna levels of rd29a ( yamaguchi - shinozaki and shinozaki , 1994 ) and chs ( feinbaum and ausubel , 1988 ), which are down - regulated by abf2 under normal condition but up - regulated under high salt condition , were higher in 35s - aria plants . on the other hand , sus ] ( martin et al ., 1993 ) and adh1 ( de bruxelles et al ., 1996 ) expression levels , which are down - regulated by abf2 under normal condition , were slightly lower than wild type levels . in aria mutant plants , chs rna level was reduced , whereas sus ] rna level was elevated , further suggesting the regulatory role of aria in their expression . thus , over - or under - expression of aria altered the expression of several abf2 - regulated genes , suggesting that it may be involved in the abf2 - dependent gene regulation process . we described an arm repeat protein designated as aria , which specifically interacts with abf2 . in animals , arm proteins are involved in a variety of cellular functions such as cell - contact , signal transduction , tumor suppression , and nuclear import ( hatzfeld , 1999 ; andrade et al ., 2001 ). in plants , their functions are largely unknown except for a few genes . arc1 and phor1 are regulators of self - incompatibility and ga signaling , respectively , as mentioned earlier ( stone et al ., 1999 ; amador et al ., 2001 ). a tobacco arm repeat protein , ntpub4 , which interacts with a receptor - like kinase , has been suggested to be a developmental regulator ( kim et al ., 2003 ). the majority ( approximately 40 %) of arabidopsis arm repeat proteins , including arc1 and phor1 , contain the u - box motif found in a subclass of e3 unbiquitin ligases ( coates , 2003 ; mudgil et al ., 2004 ). many of them therefore may participate in the ubiquitin - dependent protein degradation process . aria , however , does not contain the u - box motif . instead , it possesses another conserved sequence motif , btb / poz domain . sequence comparison indicates that only two arabidopsis proteins , including aria , have both arm repeat and btb / poz domain . the btb / poz domain is found in many transcription factors and in some actin - binding proteins in animals ( aravind and koonin , 1998 ; collins et al ., 2001 ). most recent studies show that some btb domain proteins are substrate - specific adapters for the cul - 3 - based e3 ubiquitin ligases ( van den heuvel , 2004 ). although arm repeat and btb domain proteins play diverse roles , the basic functions of the two motifs are to mediate protein - protein interactions . thus , aria has the potential to form complexes with other proteins or to function as a scaffold . the physiological relevance of the abf2 - aria interaction was supported by their similar expression patterns . expression of both abf2 and aria is induced by aba and high salt ( fig2 ) ( choi et al ., 2000 ). both are highly expressed in vegetative tissues ( especially , in lateral roots , leaf vascular tissues , and guard cells ) and in reproductive organs ( i . e ., anthers , filaments , stigma , and abscission zone ). furthermore , both proteins are localized in the nucleus ( fig2 ), although aria is also found in the cell membrane or cell wall region . our data on the in vivo function of aria further support the physiological significance of the abf2 - aria interaction . aria overexpression enhanced aba and osmolarity sensitivities at the germination stage . during subsequent seedling growth , it enhanced salt tolerance . disruption of its expression , on the other hand , promoted germination / seedling growth and impaired glucose response . several of the 35s - aria and aria mutant phenotypes are similar to those of 35s - abf2 and abj2 plants . for instance , delayed germination of overexpression lines , faster germination / growth of mutant seedlings , salt tolerance of overexpression lines , and glucose insensitivity of knockout mutants were also observed with abf2 ( kim et al ., 2004 ). furthermore , we observe altered expression of several abf2 - responsive genes in 35s - aria and aria plants ( fig5 ), suggesting that aria affects abf2 - regulated gene expression . our results indicate that aria is a positive component of aba signaling . aba sensitivity was enhanced by its overexpression and impaired by its knockout mutation . germination was delayed by its overexpression and promoted by its mutation . also , other aba - associated processes such as osmolarity sensitivity and sugar response were positively and negatively affected by aria overexpression and its mutation , respectively . two observations are worthy to be mentioned regarding the role of aria in aba response . first , most of the aria overexpression and knockout phenotypes are relatively weak or partial ( fig3 and 4 ), although they are consistently observed . this implies that the function of aria might be redundant . as mentioned before , there is an arm repeat / btb domain protein in the arabidopsis genome , which is highly homologous to aria not only in the amino acid sequence but also in its gene structure ( data not shown ), and , thus , functional redundancy between the two proteins can be speculated . another observation is that aria affects only a subset of aba - dependent processes . aba sensitivity during germination and young seedling growth was affected by aria . however , other aba - dependent processes , such as stomatal closure and abiotic stress responses other than salt tolerance , were not significantly affected by it ( data not shown ). the altered expression of several abf2 - regulated genes ( fig5 ) suggests that aria affects the abf2 - dependent gene expression . we do not know the biochemical mechanism of aria function at present . however , it can be speculated that it may function as a coactivator or repressor of abf2 . in animals , the arm protein , β - catenin , has been demonstrated to be a transcriptional coactivator ; i . e ., it translocates into the nucleus in response to a hormone signal and form complexes with transcription factors to activate target gene expression ( polakis , 2000 ). the btb / poz domain , on the other hands , is known to mediate transcriptional repression by recruiting transcriptional corepressors , which , in turn , recruit histone deacetylase to suppress transcription ( collins et al ., 2001 ). the btb / poz domain is also involved in protein degradation ( van den heuvel , 2004 ). thus , aria might be involved in the stability control of abf2 or other proteins that possibly might associate with it . since aria possesses two protein - protein interaction domains , another possibility is that it may function as an adaptor for abf2 to form a protein complex . whatever the biochemical mechanism ( s ) of aria function may be , our data indicate that overexpression of aria enhances the salt tolerance of arabidopsis plants . the result suggests that expression of aria can be engineered to promote the salt tolerance of plants , and thus it will be possible to develop salt - tolerant plants utilizing the aria gene . dna manipulation and rna gel blot analyses were performed according to the standard methods ( sambrook and russel , 2001 ). dna sequencing was done on abi 310 genetic analyzer ( applied biosystems ). rna was isolated by the method of chomczynski and mackey ( 1995 ) and purified further by licl precipitation and ethanol precipitation . aba , salt , cold , and drought treatments of arabidopsis seedlings were conducted as described ( choi et al ., 2000 ). for rna gel blot analysis , 25 μg of total rna was separated on 1 . 1 % formaldehyde agarose gel , transferred to nylon membrane ( hybond - xl , amersham pharmacia biotech ), and fixed using stratagene &# 39 ; s uv crosslinker ( model 2400 ). hybridization was carried out at 65 c for 18 - 24 hr in rapid - hyb buffer ( amersham pharmacia biotech ), using a 32 p - labelled dna fragment containing the less - well conserved region ( amino acid position 336 - 554 ) of aria as a probe . filters were washed sequentially as follows : twice in 2 × ssc ( 1 × ssc is 0 . 15 m nacl , 0 . 015 m sodium citrate ) for 10 min at room temperature , twice in 0 . 2 × ssc for 10 min at room temperature , twice in 0 . 2 × ssc for 10 min at 65 ° c . exposure was done at − 70 ° c . rt - pcr was carried out by processing 0 . 5 μg of total rna according to the manufacturer &# 39 ; s instruction , employing the access rt - pcr system ( promega ). primer sets , including the actin primers used for control reaction ( arabidopsis actin - 1 gene , accession number m20016 ), were described previously ( kang et al ., 2002 ). rna samples were confirmed to be free of contaminating dna by using the actin primer set that spans an intron and , when possible , also by using primer sets spanning an intron ( s ). the number of pcr cycles was variable depending on specific genes ( generally 20 - 30 cycles ), within the linear range of pcr amplification . the results of rt - pcr were confirmed by several independent reactions . yeast growth and transformation were according to the standard techniques ( guthrie and fink , 1991 ). two - hybrid screens were carried out employing the matchmaker lexa two - hybrid system ( clontech ), with some modifications . bait constructs were prepared by cloning two partial fragments of abf2 into pgilda ( clontech ), which carries the lexa dna binding domain under the control of the gal1 promoter and the his3 marker gene . the abf2 fragments , spanning amino acid residues 65 - 162 ( conserved region ) and 234 - 337 ( variable region ), respectively , were prepared by pcr ( primer sets , 5 ′- gctagtggtgtggttccagtt c - 3 ′ ( seq id no : 3 ) and 5 ′- gagagctcgagctgagctcttgcagcaacctg - 3 ′ ( seq id no : 4 ), and 5 ′- ccaatcatgcctaagcagcc - 3 ′ ( seq id no : 5 ) and 5 ′- gagagctcgagctctacaac tttctccacagtg - 3 ′ ( seq id no : 6 ), respectively ), and , after digestion with xho i , ligated with pgilda , which in turn was prepared by bam hi digestion , klenow fill - in reaction , and xho i digestion . the bait constructs were then individually introduced into the reporter yeast , egy48 ( matα , his3 , trp1 , ura3 :: lexa op ( x8 ) - lacz , lexa op ( x6 ) - leu2 ), by transformation . the egy48 strain carries two reporter genes , leu2 and lacz , integrated into the chromosome . large - scale transformation for the screening was carried out as described ( choi et al ., 2000 ). the reporter yeast was transformed with library plasmid dna representing cdna of aba / salt - treated arabidopsis seedlings ( choi et al ., 2000 ). transformed yeast was grown on gal / raf / cm - his - leu - trp - ura medium for 5 - 7 days , and positive colonies were identified by colony lift β - galactosidase assay . the leu + / lacz + positive colonies were purified by streaking on the same selection medium followed by another round of β - galactosidase assay . for each reporter yeast , 6 . 6 million transformants were screened , and five positive clones were obtained from the variable region bait , whereas no positive clones were obtained from the conserved region bait . specificity of the interaction of the positive clones was tested by re - transforming the reporter yeast with the plasmid dna rescued from the clones ( see below ). plasmid rescue and insert dna analysis were carried out as described ( choi et al ., 2000 ). sequencing of the plasmid dna rescued from the positive clones revealed that three of them ( clones 12 , 20 , and 24 ) encoded an arm repeat protein ( at5g19330 ) and two of them ( clones 17 and 27 ) encoded a transcription factor . the longest arm protein clone was missing the first five amino acid residues . full - length gene was isolated by pcr using the primer set , 5 ′- ggatcgtcttttactttgtgaacg - 3 ′ ( seq id no : 7 ) and 5 ′- cattcaa gac cga ttg tgatcag - 3 ′ ( seq id no : 8 ), and 1 μg of library dna . the pcr product , which contains the entire coding region and 5 ′ ( 208 bases ) and 3 ′ ( 24 bases ) additional sequences , was cloned into the zero blunt ® topo pcr cloning kit ( invitrogen ) and sequenced fully . the correctness of its nucleotide sequence was confirmed by comparing it with the genomic sequence on the arabidopsis database . gst - aria fusion constructs were prepared by cloning pcr fragments of various portions ( full - length , amino acids 1 - 518 , and amino acids 511 - 710 ) of aria into the sma i site of pgex - 6p - 2 ( amersham pharmacia biotech ). constructs were used to transform bl21 cells , and transformed cells were grown in 2 × yt medium containing 50 μg / ml ampicillin overnight . the cultures were diluted 100 - fold and grown to a 600 of 0 . 6 at 30 ° c . ( btb construct ) or 37 ° c . ( full - length and arm constructs ). the expression of recombinant proteins was induced with 0 . 5 mm isopropyl - β - d - thiogalactopyranoside for 3 hr . at the end of the induction , cells were pelleted down by centrifugation , resuspended in 6 ml of pbs ( 0 . 14 m nacl , 2 . 7 mm kcl , 10 . 1 mm na 2 hpo 4 , 1 . 8 mm kh 2 po 4 , ph7 . 3 ), and sonicated . the lysate was cleared of cell debris by centrifugation and further purified according to the supplier &# 39 ; s instruction . for in vitro translation of abf2 , full - length abf2 cloned into pcite ( novagen ), was processed with the tnt ® in vitro translation kit ( promega ) in the presence of 35 s - met according to the manufacturer &# 39 ; s instruction . for binding assay , gst - aria fusion proteins ( 0 . 5 μg ) were incubated with the glutathione - sepharose 4b resins for 1 hr at 4 ° c . in a binding buffer ( 50 mm tris , ph 8 . 0 , 100 mm nacl , 10 % glycerol , 0 . 5 % triton x - 100 , 1 mm pmsf ). in vitro - translated , 35 s - labeled abf2 was then added and incubation was continued for 2 hr with constant rotation . the resins were washed five times with the binding buffer and resuspended in sds - polyacrylamide gel electrophoresis sample buffer . the proteins were separated on 15 % sds - polyacrylamide gel and visualized by autoradiography . a 2 . 1 kb promoter fragment was prepared by pcr , using the primer set , 5 ′- gatccgaag aagaggagagatc - 3 ′ ( seq id no : 9 ) and 5 - gccacgctgtcttctttcactacact aaa aaatacagc - 3 ′ ( seq id no : 10 ), and cloned into the hind iii - xba i sites of pbi101 . 2 . the construct was introduced into arabidopsis ( ler ) by transformation , and t2 or t3 generation plants were used for the analysis of gus activity . gus staining was performed according to jefferson et al . ( 1987 ). whole plants or tissues were immersed in 1 mm 5 - bromo - 4 - chloro - 3 - indolyl - β - glucuronic acid ( x - gluc ) solution in 100 mm sodium phosphate , ph 7 . 0 , 0 . 1 mm edta , 0 . 5 mm ferricyanide , 0 . 5 mm ferrocyanide , and 0 . 1 % triton x - 100 for 24 hr at 37 ° c . chlorophyll was cleared from the tissues by ethanol series : 35 %, 50 %, and 70 %. to prepare the 35s - aria - gfp fusion construct , the entire coding region of aria was prepared by pcr , and after digestion with nco i - spe i , cloned into the same sites of pcambia1302 ( cambia ). the construct was introduced into arabidopsis ( col - 0 ) by transformation , and t1 plants were used for gfp localization analysis . nuclei were visualized by propidium iodide ( pi )- staining . roots of 10 - day - old transgenic seedlings were used for the green ( gfp localization ) and red ( pi ) fluorescence analysis using a confocal microscope ( leica , tcs - nt ). to investigate abf2 localization , the coding region of abf2 was inserted in front of the gus coding region of pbi221 in frame . onion epidermal cells were then transiently transformed with the abf2 - gus construct by particle bombardment using pds 1000 ( bio - rad ). gus activity was determined by x - gluc ( 5 - bromo - 4 - chloro - 3 - indolyl - β - glucuronic acid ) staining after 24 hr at 23 ° c . nuclei were visualized by 4 ′, 6 - diamidino - 2 - phenylindole ( dapi ) staining and observed under a fluorescence microscope ( olympus bx51 ). to prepare the 35s - aria construct , the coding region of aria was prepared by pcr , using primers 5 ′- cgcggatccatggaccaacaaccggagagg - 3 ′ ( seq id no : 11 ) and 5 ′- gcgggatcc caacctcaag ctttg caggtt tg - 3 ′ ( seq id no : 12 ), and , after digestion with bam hi , cloned into the bam hi site of pbii21 lacking the gus coding region . transformation of arabidopsis ( ler ) was according to the vacuum infiltration method ( bechtold and pelletier , 1998 ), using a . tumefaciens strain gv3 101 . seven homozygous lines were recovered and , after preliminary analysis , two representative lines ( t4 ) were chosen for detailed analysis . to establish aria mutant lines , four putative aria knockout mutant lines were obtained from the arabidopsis stock center . the stock seeds were sown and grown on soil , and seeds were harvested from individual plants . to choose t - dna insertion lines with single integration , segregation ratio of kanamycin resistancy ( kan r ) was tested , and homozygous sublines were established from those segregating at 3 : 1 ratio of kan r : kan s . genomic dna was isolated from the sublines and the integration of t - dna at the annotated site was confirmed by the sequencing of pcr fragments . we were able to identify one insertion line ( salk — 143439 ) with a single t - dna insertion at the annotated site among the four putative lines . t - dna is inserted at − 379 from the translation start site . expression analysis by rt - pcr showed that aria expression is abolished in the insertion line . for phenotype analysis , two sublines ( ark5 and ark10 ) were used . same results were obtained from them and those from ar10 are presented . arabidopsis thaliana ecotypes landsberg erecta ( ler ) and columbia ( col - 0 ) were used . plants were grown under long day condition ( 16 hr light / 8 hr dark cycle ) at 22 ° c ., on 1 : 1 : 1 mixture of vermiculite , perlite and peat moss or on ms plates . soil - 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