BACKGROUND
tor, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation  <cit> .
tor is a large  protein kinase  <cit>  present in all eukaryotes analyzed.
it is thought to act in a nutrient-sensitive complex torc <dig> with raptor  and another protein to regulate cell growth  <cit>  – though there is some debate on the nutrient sensitivity of the complex  <cit> .
raptor, a protein with heat and wd- <dig> protein interaction domains, recruits substrates for phosphorylation by tor in yeast and mammals  <cit> .
tor also acts in a second, nutrient-insensitive complex without raptor to regulate the cytoskeleton  <cit> .
disruption of the arabidopsis tor homologue attor is lethal early in plant embryonic development  <cit> .
disruption of atraptor  causes seedling developmental arrest but allows normal embryonic development , suggesting that attor embryonic activity is independent of atraptor and that the tor-raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth.
in support of this, attor has been show to be expressed in dividing and expanding cells  <cit> .
thus, identifying downstream activators of tor signaling may provide insights into the activity of attor in post-embryonic growth.
mei <dig> is a putative tor substrate and potent meiosis-signaling molecule identified in the fission yeast schizosaccharomyces pombe  <cit> .
mei <dig> is bound by the raptor homologue mip <dig>  <cit> , and is an inactive phosphoprotein under high nutrient conditions  <cit>  – conditions which promote tor kinase activity.
the kinase governing two of the three mei <dig> phosphorylation sites is known  <cit> ; tor is a strong candidate for the kinase governing the third.
in diploids under low nutrient conditions, unphosphorylated mei <dig> accumulates and localizes to the nucleus, where it binds to a noncoding, mrna-like rna molecule meirna in an interaction mediated by the third of its three rna recognition motifs  <cit> .
the mei2-meirna interaction occurs as meirna is being transcribed, tethering mei <dig> to the meirna locus  <cit> .
accumulation of mei <dig> at this focused point immediately precedes meiosis.
mei2-like proteins are predicted in a wide range of organisms  <cit>  including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast.
in land plants, predicted mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues  <cit> .
arabidopsis mei2-like  <dig> , the first member of this family to be described, was isolated in a screen for plant cdnas whose expression could complement defects in the fission yeast meiosis signaling pathway  <cit> .
like mei <dig>  aml <dig> has three rrms.
expression of a protein fragment containing only the third aml <dig> rrm was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of mei <dig>  but not in lesions of the mei <dig> locus itself.
terminal ear  <dig> , a more divergent member of the mei2-like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear  <cit> .
given the potential role of downstream attor effectors in post-embryonic growth, the intriguing signaling activity of mei <dig> in fission yeast, and the known interaction between mei <dig> and the fission yeast raptor protein, we asked if the mei2-raptor interaction was conserved between its arabidopsis orthologues aml <dig> and atraptor1b.
RESULTS
aml <dig> and atraptor1b interact in a yeast two-hybrid assay
to test for an interaction between the arabidopsis proteins aml <dig> and atraptor1b, the open reading frames  of each of the transcripts predicted to encode these proteins were amplified via pcr with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pgadt <dig> and pgbkt <dig>  pgadt <dig> encodes a transcriptional activation domain  which can recruit the yeast transcription machinery.
pgbkt <dig> encodes a dna binding-domain  which binds to the promoters of ade <dig> and his <dig>  pgadt <dig> and pgbkt <dig> additionally carry the genes leu <dig> and trp <dig>  when transformed into the yeast line ah <dig>  which is leu2-trp1-ade2-his3-, co-transformed cells will grow on yeast medium lacking leucine and tryptophan.
cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine .
ah <dig> yeast cells co-transformed with clones encoding aml <dig> and atraptor1b in complementary two-hybrid vectors grew on selective media.
co-transformations of control pgbkt <dig> and pgadt <dig> empty vectors, or either control vector co-transformed with its complement harboring the aml <dig> or atraptor1b orf, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media.
evidence from other systems indicates that raptor protein fragments lose the ability to bind substrates  <cit> .
therefore, only full length atraptor1b was tested in this assay.
mei <dig>  the fission yeast aml <dig> homologue, is highly modular.
it is divided into distinct n-terminal and c-terminal domains.
the n-terminal half of the protein appears to play a regulatory role  <cit> .
the c-terminal half of mei <dig> is sufficient to complement lesions in the mei <dig> locus  <cit> .
additionally, the aml <dig> c-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of mei <dig>  <cit> .
therefore we generated clones encoding fragments of aml <dig> and assayed them for interactions with full length atraptor1b.
aml <dig> fragments n <dig> and n <dig>  comprised of the first  <dig> or the first  <dig> residues of the  <dig> residue aml <dig> protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pgadt <dig> and co-transformed with pgbk-raptor .
neither n <dig>  nor 155– <dig>  , nor 155– <dig> , could restore growth when cloned into pgadt <dig> and co-transformed with pgbk-raptor.
aml <dig> fragment 402c, comprised of residues  <dig> to the c-terminus of the protein, and all c-terminal fragments tested  failed to restore growth on selective medium.
none of the aml <dig> fragments cloned into pgadt <dig>  and cotransformed with empty pgbkt <dig> control vector, yielded transformants able to grow on selective medium.
aml <dig> fragments n <dig> and n <dig> harbor activation domain activity in a yeast one-hybrid assay
aml <dig> n <dig>  cloned into pgbkt <dig>  restored growth on selective medium to cells co-transformed with either pgad:raptor or pgadt <dig>  to investigate this result further, we tested all aml <dig> fragments for native transcriptional activation domain activity in a yeast one-hybrid assay.
aml <dig> fragments were cloned into pgbkt <dig>  singly transformed into ah <dig> yeast cells, and assayed for growth on media lacking tryptophan  and media additionally lacking adenine.
the dna binding-domain of the pgbkt <dig> tethers any c-terminally fused fragments to the ade <dig> promoter.
in the absence of a binding partner, bd-fusion chimeric proteins trigger transcription of ade <dig> only if the protein fused to the bd contains native transcriptional activation activity.
aml <dig> n <dig> and aml <dig> n <dig>  but not full length aml <dig>  aml <dig> n <dig> or any c-terminal aml <dig> fragments, were able to restore growth on media lacking tryptophan and adenine .
aml <dig> n <dig> was similarly unable to restore growth.
aml <dig> fragments 155– <dig> and 155– <dig>  in pgbkt <dig>  could not be stably transformed into yeast.
these results were observed in multiple independent transformant lines for a given construct.
cotransformation of any of the pgbkt7-derived constructs with pgadt <dig> did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine.
plants homozygous for insertion alleles of aml <dig> and of all five aml family members show early flowering
an interaction with atraptor1b points to aml <dig> as a downstream effector of tor signaling in plants.
additionally, the dramatic phenotype mei <dig> disruption and the intriguing mode of mei <dig> action led us to ask what the consequences would be of disruption of the aml <dig> locus and of all five aml gene family members.
to obtain insertion alleles in aml <dig> and other aml gene family members, we screened the insertion allele populations at the university of wisconsin arabidopsis knockout facility  <cit> , obtaining alleles harboring insertions in aml <dig>  aml <dig>  aml <dig> and aml <dig> .
an aml <dig> insertion allele was obtained from the signal collection at the salk institute  <cit> .
by rt-pcr using primers which anneal to the cdna at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants .
by a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes q <dig> and q <dig>  all lines were viable and fertile.
aml insertion homozygotes bolted earlier than wild-type lines .
this effect was independent of the number of insertion alleles carried by the mutants; aml 'quint' lines q <dig> and q <dig> were not qualitatively different than lower order insertion allele homozygotes.
additionally, aml insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules.
seedlings were germinated on culture medium supplemented with the gibberellic acid ga <dig>  paclobutrazol, the auxin  <dig> -d, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark.
quintuple insertion mutants responded slightly more than wild-type seedlings to ga <dig> as measured by the change in seedling length in the presence vs. in the absence of the hormone.
this effect was repeatable but weak, and no other differential hormonal response was observed .
sequence downstream of the mutant allele insertion sites is transcribed
given the mild phenotype of the aml insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus.
rt-pcr, as previously stated, showed that no wild-type transcripts accumulate in lines q <dig> and q <dig>  in fission yeast, however, the c-terminal half of the protein is sufficient to complement lesions of the mei <dig> locus  <cit> .
the insertion alleles of all but aml <dig> are disrupted at or near the 5' ends of their predicted coding regions.
we therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the c-terminal half of any of the aml proteins .
to determine whether the amplified fragments corresponded to aml cdnas, we performed restriction digests on the pcr products, which confirmed that the cdnas originated from aml gene transcripts.
weak amplification of cdna representing transcripts originating downstream of the insertion site was observed for aml <dig>  aml <dig> and aml3; amplification of the aml <dig> 3' region was indistinguishable from the amplification seen from of wild-type cdna template.
transgenic lines overexpressing aml1:gfp or gfp:aml <dig> fusion proteins could not be recovered
the aml <dig> orf was separately cloned into the pcambia <dig> 35s::gfp plant transformation vector both 5' and 3' of the gfp orf, and the construct was transformed into arabidopsis via agrobacterium-mediated floral dip  <cit> .
transformants, identified by resistance to the antibiotic hygromycin and confirmed through pcr, were recovered at a very low rate of less than .01%.
no fluorescence was observed in any tissues of any transformants assayed, and aml1:gfp transcripts could not be detected via rt-pcr performed on cdna transcribed from rna extracted from bulk shoot tissue .
discussion
raptor proteins in yeast and mammals function by recruiting substrates for tor, a central regulator of cell growth in response to nutrients  <cit> .
an interaction with raptor therefore strongly suggests that a given protein is a tor substrate and downstream effector of tor signaling.
plants homozygous for lesions at both atraptor loci show normal embryonic development but are unable to maintain shoot meristem activity  <cit> .
tor substrates, then, may play a role in regulating meristem-driven post-embryonic growth.
the interaction between aml <dig> and atraptor1b implicates the aml family of proteins in tor signaling.
it points specifically to a role for the aml proteins in regulation of shoot meristem activity.
an interaction between mei <dig> and the fission yeast raptor homologue mip <dig> has been reported previously; indeed, mip <dig>  was the first raptor homologue characterized in any eukaryote  <cit> .
the conservation of this interaction from fission yeast to plants suggests that the well-characterized mei <dig> signaling pathway may provide insight into the function of the amls.
mei <dig> is a potent meiosis-signaling molecule.
it triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress  <cit> .
meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues.
thus the amls may also play a role in cellular differentiation or in meiosis signaling.
aside from the effect of mei <dig> in development, there is the intriguing issue of its mode of action.
mei <dig> sub-cellular localization is mediated by an interaction with a noncoding, mrna-like molecule  <cit> .
there is a fairly large population of mrna-like transcripts conserved among land plants despite lacking large conserved open reading frames  <cit> .
of these, the conserved alfalfa transcript enod <dig> has been shown to mediate the sub-cellular localization of an rna-binding protein  <cit>  and to mediate phytohormone responses  <cit> .
aml <dig> may be a binding partner of one or more of these mrna-like noncoding molecules in plants.
the transcriptional activation activity of the aml <dig> n-terminus observed in the yeast one hybrid assay has not been ascribed to mei <dig> and may represent a novel activity of plant mei2-like proteins.
this activation activity localizes to the n-terminal  <dig> residues, but is strongly influenced by the adjacent residues.
activity is lost in n <dig>  regained on n <dig> and lost again in full length aml <dig>  this suggests that the aml <dig> n-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations.
we should emphasize, however, that the aml <dig> n-terminal fragment transcriptional activation activity has yet to be shown in planta, and that the activity may result from fusion to the dna binding domain rather than being present in native aml <dig>
atraptor1b binding to aml <dig> is also localized to the n-terminus, and appears to be mediated by multiple sites in this region.
this suggests that the n-terminus may contain a tor phosphorylation site, and that this site may influence the configuration of the n-terminus.
the repeated failure to recover transgenic lines expressing aml <dig> suggests that its unregulated overexpression is lethal.
future efforts to characterize the aml proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated aml expression.
disruption of any of the aml loci causes early bolting in plants grown under long days.
however, lines homozygous for insertions in all five aml loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that rt-pcr performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate.
transcripts originating downstream of the insertion sites but still capable of encoding the c-terminal half of the wild-type protein accumulate from all loci but aml <dig>  this raises the possibility that the aml quintuple insertion homozygote lines do not represent total disruption of aml activity.
four of the five aml open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity  remains and may be sufficient to cause transcription of aml coding region dna downstream of the insertion site.
viewed in this light, the early flowering phenotype of aml insertion homozygotes may arise not from the total disruption of aml activity but from the accumulation of aml proteins which, due to the truncations in their n-termini caused by the insertions, are no longer bound by atraptor1b, no longer phosphorylated by attor, or no longer able to activate transcription of floral repressors.
finally, these results provide a cautionary tale.
rt-pcr performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.
CONCLUSIONS
tor is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants.
this work shows that aml <dig> binds atraptor1b, and suggests that the aml protein family may be phosphorylated by attor in an atraptor1b-mediated interaction.
the interaction with atraptor1b implicates aml <dig> as a downstream effector of attor kinase signaling, and provides insight into the mode of action of this critical growth regulator.

 tor, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation. tor is a large protein kinase present in all eukaryotes analyzed. it is thought to act in a nutrient-sensitive complex torc with raptor and another protein to regulate cell growth – though there is some debate on the nutrient sensitivity of the complex. raptor, a protein with heat and wd- protein interaction domains, recruits substrates for phosphorylation by tor in yeast and mammals. tor also acts in a second, nutrient-insensitive complex without raptor to regulate the cytoskeleton. disruption of the arabidopsis tor homologue attor is lethal early in plant embryonic development. disruption of atraptor causes seedling developmental arrest but allows normal embryonic development, suggesting that attor embryonic activity is independent of atraptor and that the tor-raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth. in support of this, attor has been show to be expressed in dividing and expanding cells. thus, identifying downstream activators of tor signaling may provide insights into the activity of attor in post-embryonic growth. mei is a putative tor substrate and potent meiosis-signaling molecule identified in the fission yeast schizosaccharomyces pombe. mei is bound by the raptor homologue mip, and is an inactive phosphoprotein under high nutrient conditions – conditions which promote tor kinase activity. the kinase governing two of the three mei phosphorylation sites is known; tor is a strong candidate for the kinase governing the third. in diploids under low nutrient conditions, unphosphorylated mei accumulates and localizes to the nucleus, where it binds to a noncoding, mrna-like rna molecule meirna in an interaction mediated by the third of its three rna recognition motifs. the mei2-meirna interaction occurs as meirna is being transcribed, tethering mei to the meirna locus. accumulation of mei at this focused point immediately precedes meiosis. mei2-like proteins are predicted in a wide range of organisms including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast. in land plants, predicted mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues. arabidopsis mei2-like, the first member of this family to be described, was isolated in a screen for plant cdnas whose expression could complement defects in the fission yeast meiosis signaling pathway. like mei aml has three rrms. expression of a protein fragment containing only the third aml rrm was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of mei but not in lesions of the mei locus itself. terminal ear, a more divergent member of the mei2-like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear. given the potential role of downstream attor effectors in post-embryonic growth, the intriguing signaling activity of mei in fission yeast, and the known interaction between mei and the fission yeast raptor protein, we asked if the mei2-raptor interaction was conserved between its arabidopsis orthologues aml and atraptor1b.  aml and atraptor1b interact in a yeast two-hybrid assay to test for an interaction between the arabidopsis proteins aml and atraptor1b, the open reading frames of each of the transcripts predicted to encode these proteins were amplified via pcr with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pgadt and pgbkt pgadt encodes a transcriptional activation domain which can recruit the yeast transcription machinery. pgbkt encodes a dna binding-domain which binds to the promoters of ade and his pgadt and pgbkt additionally carry the genes leu and trp when transformed into the yeast line ah which is leu2-trp1-ade2-his3-, co-transformed cells will grow on yeast medium lacking leucine and tryptophan. cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine. ah yeast cells co-transformed with clones encoding aml and atraptor1b in complementary two-hybrid vectors grew on selective media. co-transformations of control pgbkt and pgadt empty vectors, or either control vector co-transformed with its complement harboring the aml or atraptor1b orf, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media. evidence from other systems indicates that raptor protein fragments lose the ability to bind substrates. therefore, only full length atraptor1b was tested in this assay. mei the fission yeast aml homologue, is highly modular. it is divided into distinct n-terminal and c-terminal domains. the n-terminal half of the protein appears to play a regulatory role. the c-terminal half of mei is sufficient to complement lesions in the mei locus. additionally, the aml c-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of mei. therefore we generated clones encoding fragments of aml and assayed them for interactions with full length atraptor1b. aml fragments n and n comprised of the first or the first residues of the residue aml protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pgadt and co-transformed with pgbk-raptor. neither n nor 155–, nor 155–, could restore growth when cloned into pgadt and co-transformed with pgbk-raptor. aml fragment 402c, comprised of residues to the c-terminus of the protein, and all c-terminal fragments tested failed to restore growth on selective medium. none of the aml fragments cloned into pgadt and cotransformed with empty pgbkt control vector, yielded transformants able to grow on selective medium. aml fragments n and n harbor activation domain activity in a yeast one-hybrid assay aml n cloned into pgbkt restored growth on selective medium to cells co-transformed with either pgad:raptor or pgadt to investigate this result further, we tested all aml fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. aml fragments were cloned into pgbkt singly transformed into ah yeast cells, and assayed for growth on media lacking tryptophan and media additionally lacking adenine. the dna binding-domain of the pgbkt tethers any c-terminally fused fragments to the ade promoter. in the absence of a binding partner, bd-fusion chimeric proteins trigger transcription of ade only if the protein fused to the bd contains native transcriptional activation activity. aml n and aml n but not full length aml aml n or any c-terminal aml fragments, were able to restore growth on media lacking tryptophan and adenine. aml n was similarly unable to restore growth. aml fragments 155– and 155– in pgbkt could not be stably transformed into yeast. these results were observed in multiple independent transformant lines for a given construct. cotransformation of any of the pgbkt7-derived constructs with pgadt did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine. plants homozygous for insertion alleles of aml and of all five aml family members show early flowering an interaction with atraptor1b points to aml as a downstream effector of tor signaling in plants. additionally, the dramatic phenotype mei disruption and the intriguing mode of mei action led us to ask what the consequences would be of disruption of the aml locus and of all five aml gene family members. to obtain insertion alleles in aml and other aml gene family members, we screened the insertion allele populations at the university of wisconsin arabidopsis knockout facility, obtaining alleles harboring insertions in aml aml aml and aml. an aml insertion allele was obtained from the signal collection at the salk institute. by rt-pcr using primers which anneal to the cdna at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants. by a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes q and q all lines were viable and fertile. aml insertion homozygotes bolted earlier than wild-type lines. this effect was independent of the number of insertion alleles carried by the mutants; aml'quint' lines q and q were not qualitatively different than lower order insertion allele homozygotes. additionally, aml insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. seedlings were germinated on culture medium supplemented with the gibberellic acid ga paclobutrazol, the auxin -d, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. quintuple insertion mutants responded slightly more than wild-type seedlings to ga as measured by the change in seedling length in the presence vs. in the absence of the hormone. this effect was repeatable but weak, and no other differential hormonal response was observed. sequence downstream of the mutant allele insertion sites is transcribed given the mild phenotype of the aml insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. rt-pcr, as previously stated, showed that no wild-type transcripts accumulate in lines q and q in fission yeast, however, the c-terminal half of the protein is sufficient to complement lesions of the mei locus. the insertion alleles of all but aml are disrupted at or near the 5' ends of their predicted coding regions. we therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the c-terminal half of any of the aml proteins. to determine whether the amplified fragments corresponded to aml cdnas, we performed restriction digests on the pcr products, which confirmed that the cdnas originated from aml gene transcripts. weak amplification of cdna representing transcripts originating downstream of the insertion site was observed for aml aml and aml3; amplification of the aml 3' region was indistinguishable from the amplification seen from of wild-type cdna template. transgenic lines overexpressing aml1:gfp or gfp:aml fusion proteins could not be recovered the aml orf was separately cloned into the pcambia 35s::gfp plant transformation vector both 5' and 3' of the gfp orf, and the construct was transformed into arabidopsis via agrobacterium-mediated floral dip. transformants, identified by resistance to the antibiotic hygromycin and confirmed through pcr, were recovered at a very low rate of less than.01%. no fluorescence was observed in any tissues of any transformants assayed, and aml1:gfp transcripts could not be detected via rt-pcr performed on cdna transcribed from rna extracted from bulk shoot tissue. discussion raptor proteins in yeast and mammals function by recruiting substrates for tor, a central regulator of cell growth in response to nutrients. an interaction with raptor therefore strongly suggests that a given protein is a tor substrate and downstream effector of tor signaling. plants homozygous for lesions at both atraptor loci show normal embryonic development but are unable to maintain shoot meristem activity. tor substrates, then, may play a role in regulating meristem-driven post-embryonic growth. the interaction between aml and atraptor1b implicates the aml family of proteins in tor signaling. it points specifically to a role for the aml proteins in regulation of shoot meristem activity. an interaction between mei and the fission yeast raptor homologue mip has been reported previously; indeed, mip was the first raptor homologue characterized in any eukaryote. the conservation of this interaction from fission yeast to plants suggests that the well-characterized mei signaling pathway may provide insight into the function of the amls. mei is a potent meiosis-signaling molecule. it triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress. meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues. thus the amls may also play a role in cellular differentiation or in meiosis signaling. aside from the effect of mei in development, there is the intriguing issue of its mode of action. mei sub-cellular localization is mediated by an interaction with a noncoding, mrna-like molecule. there is a fairly large population of mrna-like transcripts conserved among land plants despite lacking large conserved open reading frames. of these, the conserved alfalfa transcript enod has been shown to mediate the sub-cellular localization of an rna-binding protein and to mediate phytohormone responses. aml may be a binding partner of one or more of these mrna-like noncoding molecules in plants. the transcriptional activation activity of the aml n-terminus observed in the yeast one hybrid assay has not been ascribed to mei and may represent a novel activity of plant mei2-like proteins. this activation activity localizes to the n-terminal residues, but is strongly influenced by the adjacent residues. activity is lost in n regained on n and lost again in full length aml this suggests that the aml n-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations. we should emphasize, however, that the aml n-terminal fragment transcriptional activation activity has yet to be shown in planta, and that the activity may result from fusion to the dna binding domain rather than being present in native aml atraptor1b binding to aml is also localized to the n-terminus, and appears to be mediated by multiple sites in this region. this suggests that the n-terminus may contain a tor phosphorylation site, and that this site may influence the configuration of the n-terminus. the repeated failure to recover transgenic lines expressing aml suggests that its unregulated overexpression is lethal. future efforts to characterize the aml proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated aml expression. disruption of any of the aml loci causes early bolting in plants grown under long days. however, lines homozygous for insertions in all five aml loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that rt-pcr performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate. transcripts originating downstream of the insertion sites but still capable of encoding the c-terminal half of the wild-type protein accumulate from all loci but aml this raises the possibility that the aml quintuple insertion homozygote lines do not represent total disruption of aml activity. four of the five aml open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity remains and may be sufficient to cause transcription of aml coding region dna downstream of the insertion site. viewed in this light, the early flowering phenotype of aml insertion homozygotes may arise not from the total disruption of aml activity but from the accumulation of aml proteins which, due to the truncations in their n-termini caused by the insertions, are no longer bound by atraptor1b, no longer phosphorylated by attor, or no longer able to activate transcription of floral repressors. finally, these results provide a cautionary tale. rt-pcr performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.  tor is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. this work shows that aml binds atraptor1b, and suggests that the aml protein family may be phosphorylated by attor in an atraptor1b-mediated interaction. the interaction with atraptor1b implicates aml as a downstream effector of attor kinase signaling, and provides insight into the mode of action of this critical growth regulator. 
plants homozygous for insertion alleles at the aml locus, as well as plants homozygous for insertion alleles at all five loci in the aml gene family, bolt earlier than wild-type plants. aml interacts with atraptor1b, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator tor. atraptor1b, a plant raptor homologue, binds the aml protein in a yeast two-hybrid assay. tor, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. identification of aml as a putative downstream effector of tor gives valuable insights into the plant-specific mode of action of this critical growth regulator.
 tor, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. it acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner raptor. in arabidopsis, raptor activity is essential for postembryonic growth. though comparative studies suggest potential downstream effectors, no raptor binding partners have been described in plants.  atraptor1b, a plant raptor homologue, binds the aml protein in a yeast two-hybrid assay. this interaction is mediated by the n-terminal residues of aml and marks aml as a candidate attor kinase substrate in plants. the aml n-terminus additionally carries transcriptional activation domain activity. plants homozygous for insertion alleles at the aml locus, as well as plants homozygous for insertion alleles at all five loci in the aml gene family, bolt earlier than wild-type plants.  aml interacts with atraptor1b, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator tor. identification of aml as a putative downstream effector of tor gives valuable insights into the plant-specific mode of action of this critical growth regulator. 