BACKGROUND
plant development is remarkably plastic.
groups of totipotent cells termed meristems, which are maintained throughout the life of the plant, give rise to all post-embryonic organs from roots and leaves to petals and fruit.
this allows plants, unlike metazoans, to change their final body plans dramatically in response to environmental, hormonal and nutritional cues.
while much has been learned about the determination of cell fates in the embryo  <cit>  and the apical meristems  <cit> , less is known about the genes that control the growth that occurs in cells emerging from the plant meristem.
tor proteins were originally identified in budding yeast as the targets of rapamycin, a potent antibiotic that disrupts cell growth  <cit> .
in both yeast and metazoans, tor proteins mediate translation in response to nutrients  <cit> .
yeast tor <dig> and mammalian mtor also regulate cytoskeletal organization  <cit> .
in both yeast and mammalian cells, tor proteins form a complex, torc <dig>  with gβl  <cit>  and raptor   <cit> ; their yeast homologues are lst <dig> and kog <dig>  respectively  <cit> .
raptor contains protein-binding domains, and the raptor n-terminus shows similarity to a caspase domain  <cit> , though catalytic activity has yet to be shown.
it has been reported that the strength of the tor-raptor interaction is regulated by nutrients, though there is not yet consensus on this point  <cit> .
raptor functions in torc <dig> to recruit substrates for phosphorylation by tor; raptor binds tor substrates s6kinase and eif4e-bp, and is necessary for full tor phosphorylation of these substrates in vitro  <cit> .
the tor-raptor complex is thought to mediate the nutrient-sensitive regulation of cell growth; mutants in yeast lacking a functional complex cease cell growth in a manner that mimics rapamycin treatment  <cit> .
a second tor complex, torc <dig>  involves gβl, rictor  and perhaps other proteins  <cit> .
this complex is unaffected by rapamycin, and is thought to mediate tor cytoskeletal regulation.
in arabidopsis the single tor homologue, attor, is critical for plant development  <cit> .
attor insertion homozygote embryos undergo cell division but are unable to gain cell volume or undergo apical-basal differentiation.
although attor transcripts accumulate in all tissues assayed  <cit> , an attor-gus fusion transcript is only translated in meristematic tissue and the immediately adjacent expanding cells  <cit> .
a tor-specific microrna conserved between arabidopsis and rice  <cit>  implicates post-transcriptional regulation in attor expression.
rapamycin perturbs the activity of the nutrient-sensitive tor complex in yeast and mammals.
arabidopsis, however, is insensitive to rapamycin,  <cit>  precluding this approach to the elucidation of arabidopsis tor signaling.
here we report a characterization of atraptor1a and atraptor1b, which encode the two arabidopsis raptor homologues.
we found defects in both root and shoot growth in atraptor1b disruption lines, resulting in slow leaf initiation, late flowering and increased branching.
we also found that atraptor1a atraptor1b double mutant homozygotes undergo normal seedling development, but exhibit only minimal post-embryonic meristem activity.
we contrast these results with those found previously for the attor mutant, and discuss the implications of this work for tor signaling in plants.
RESULTS
characterization of the arabidopsis raptor homologues
to search for plant homologues of mammalian raptor, we surveyed the completed arabidopsis genome and found two loci that might encode proteins highly similar to raptor at the north ends of chromosomes  <dig> and  <dig>  we did not find an expressed sequence tag  for the first locus .
however, we used the 5'-rapid amplification of cdna ends  to establish that the locus is transcribed to an mrna with a 5' end consistent with the predicted open reading frame  of atraptor1a.
the atraptor1a protein is predicted to be  <dig>  amino acids in length.
the second locus  was represented by a single est .
we used 5' race to confirm that the est contained the full orf of atraptor1b.
the atraptor1b locus is transcribed to a  <dig>  kb mrna containing  <dig> exons, and the protein is predicted to be  <dig>  amino acids in length.
predicted atraptor1a and atraptor1b proteins show 80% identity over their entirety.
to determine the time of divergence between the two atraptor loci, we searched for raptor homologues in available plant genome data using atraptor1a and atraptor1b as query sequences.
full-length raptor loci were discovered in the available rice  and alfalfa  genome sequences.
using atraptor1b and partial cdna sequence  as guides, we determined putative protein sequences from these loci.
alignment of these sequences with atraptor1a, atraptor1b, mammalian raptor, the budding yeast raptor homologue kog <dig> and the fission yeast raptor homologue mip1p showed that there is a striking degree of conservation among all raptor homologues .
atraptor1b and the saccharomyces cereviseae raptor homologue kog <dig>  the most divergent member included in the analysis, show 28% identity throughout their length.
all plant raptor homologues encode the raptor n-terminal conserved / caspase  motif, heat repeats and wd <dig> motifs first identified in fission yeast mip <dig> and characteristic of all raptor proteins  <cit> .
from this alignment we generated a phylogeny of the raptor homologues .
the predicted atraptor proteins resolve to a single clade with a high degree of confidence, indicating that the duplication of the atraptor loci post-dates the divergence of arabidopsis from medicago, and that the loci are likely to encode functionally redundant proteins.
identification of knockout mutants in atraptor1a and atraptor1b
to gain insight into the function of the atraptor proteins, we searched for insertion alleles of each locus among the publicly available sequenced t-dna insertion lines.
insertion mutant lines salk_ <dig> and salk_ <dig>  with disruptions to the atraptor1a and atraptor1b loci, were obtained from the arabidopsis biological resource center .
lines homozygous for each insertion  were identified via polymerase chain reaction , and rna from floral buds of insertion homozygotes was used for reverse-transcriptase-pcr  to assay for accumulation of wild-type transcripts from the disrupted locus.
atraptor1a transcripts could not be detected in 1a -/- buds, but were detected in 1b -/- buds.
atraptor1b transcripts were not detected in 1b -/- buds, but were detected in 1a -/- buds.
both transcripts were detected in wild-type columbia  buds .
1b-/- seedling roots grow slowly
as a first step toward characterizing these mutant lines, col, 1a-/- and 1b-/- seedlings were germinated on culture medium.
col and 1a-/- seedlings were phenotypically indistinguishable.
however, b-/- seedlings showed distinct root growth defects: roots were thick, coiled and densely covered with hairs, and the roots had difficulty penetrating the medium .
plate-grown 1b -/- seedlings were repeatedly shorter than col or 1a-/- seedlings.
although dark grown 1b-/- seedlings were also shorter than col or 1a-/-, the difference in size was much less .
while dark-grown etiolated seedlings grow primarily through the expansion and division of embryonic hypocotyl cells, light-grown seedlings grow primarily through the production of cells from the root apical meristem.
thus the difference in size between col, 1a-/- and 1b-/- seedlings results primarily from a reduction in root apical meristem growth rather than a general defect in cell expansion or metabolism.
the roots of 1b-/- seedlings grown on 90° inclined plates were morphologically wild type but shorter than col roots.
when the plates were rotated to lay flat horizontally, new growth from the root apex of 1b-/- seedlings produced thick, coiled roots , while the previously produced root tissue remained morphologically wild type .
only those sections of the root not in contact with the medium were hairy .
thus the root growth disturbance was a defect in growth into agar rather than in gravitropism, though in the absence of resistance, roots still grew more slowly than wild type.
col and 1b-/- root apical meristems  were examined using bright-field microscopy .
1b-/- seedling rams contained all cell types present in col rams but their root morphology was much more blunt; this difference appeared to be localized to the 1b-/- zone of elongation.
1b-/- root tips showed a tendency to shed their root caps, although shedding was sometimes observed in col root tips as well.
1b-/- shoots show developmental and morphological defects
to measure developmental defects resulting from atraptor insertions, we grew col, 1a-/- and 1b-/- lines on soil, and scored their rates of leaf initiation, time to floral bud initiation, rate of cauline leaf initiation, time to flowering and number of floral shoot apices.
1b-/- plants were smaller, had a slower rate of leaf initiation, and bolted and flowered later than did col or 1a-/- plants .
mature 1b-/- plants were conspicuously bushier than col or 1a-/- homozygotes .
the primary shoot apex was shorter than that of col plants and ended prematurely in a terminal inflorescence of infertile flowers.
the growth of the plant was then taken over by axillary shoots and by secondary shoots from the basal rosette.
to quantify this phenotype, col and 1b-/- lines were grown to maturity in 16-hr days, and shoot architecture for an individual plant was scored at the shattering of the first silique.
1b-/- plants showed reduced shoot height, reduced primary stem length, increased axillary branch number and an increased number of secondary shoots compared to col and 1a-/- plants .
1b-/- branch numbers, though variable, were significantly larger than those of wild type or 1a-/-.
student's t-tests of sample pairs assuming equal variance yielded p-values of  <dig>  and  <dig>  for 1b-/- vs. col and 1b-/- vs. 1a-/- axillary branch number, respectively, and  <dig>  and  <dig>  for 1b-/- vs. col and 1b-/- vs. 1a-/- secondary shoot number.
axillary branch length did not differ significantly from col values .
this phenotype became more pronounced later in plant development and was more conspicuous under short days.
complementation of the atraptor1b-/- phenotype
to confirm that the collection of phenotypes observed in the atraptor1b-/- homozygotes was due to the mutant 1b allele, 1b-/- plants were transformed with a construct containing the atraptor1b orf and 5' utr and driven by the atraptor1b promoter.
some of these transformants showed wild-type transition to bolting, shoot branching and root growth .
we conclude that the 1b-/- phenotypes described above result from homozygosity for the lesion at the atraptor1b locus.
atraptor transcripts accumulate in dividing and expanding cell tissues
a combination of in situ rna hybridization and in silico expression analysis was used to determine the rna accumulation pattern of atraptor transcripts in arabidopsis.
the atraptor1b cdna sequence was used to generate an atraptor probe to assay transcript accumulation in the shoot tips of wild-type plants.
since atraptor1a and atraptor1b show 80% identity through the length of their transcripts, this probe likely detected expression of both loci.atraptor transcripts were detected throughout the shoot tip, in all organs of the differentiating floral bud, and deep into the differentiated inflorescence stem .
signal intensity faded with the distance from the shoot apex.
this accumulation pattern differed from that of actin , which was more prominent in dividing cells of the apex.
notably, atraptor accumulation is not restricted to the primary shoot apex.
to obtain an estimate of the relative levels of atraptor1a and atraptor1b in the signal seen in fig.
6a, atraptor1a and atraptor1b locus ids were used to query the genevestigator arabidopsis thaliana microarray database and analysis toolbox  <cit> .
atraptor1a and atraptor1b accumulate in all developmental stages .
however, atraptor1b accumulation levels are consistently fourfold higher than those of atraptor1a.
a second analysis of accumulation by tissue rather than developmental stage produced similar results.
we conclude that atraptor1a and atraptor1b show similar expression patterns.
1a-/- 1b-/- double homozygote mutant seedlings show minimal meristem growth
the high degree of similarity between atraptor1a and atraptor1b led us to suspect that they may be at least partially functionally redundant.
to eliminate all atraptor activity in a single line, 1a-/- and 1b-/- lines were crossed.
no 1a-/- 1b-/- plants could be identified among the f <dig> of this cross on soil.
phenotypically wild-type 1a-/- 1b+/- plants were identified via pcr using the primer pairs described in materials and methods and their progeny  were examined on agar plates.
of these,  <dig> seedlings  appeared wild type .
it was determined by pcr that these seedlings carried a wild-type 1b allele .
the remaining seedlings  germinated but showed minimal postembryonic shoot meristem growth .
it was determined by pcr that these seedlings were genetically 1a-/- 1b-/- .
this genotyping confirmed that the seedling arrest mutant phenotype co-segregated with homozygosity for the atraptor1b mutant allele in the atraptor1a mutant background.
we concluded that this seedling phenotype corresponded to the 1a-/- 1b-/- double mutant.
we examined 1a-/- 1b-/- double mutant seedling roots via bright field microscopy to ascertain the extent of their post-embryonic growth defect.
1a-/- 1b-/- roots were conspicuously narrower than wild type.
the columella, quiescent center, vasculature, pericycle, endodermis and cortex are present though all smaller than wild type; the outer layer of cells is difficult to identify.
further up the root, root hairs were clearly visible, indicating that the ram had produced some mature root cell types .
to gain a better understanding of the double mutant shoot apical meristem  defect, col and 1a-/- 1b-/- 7-day seedlings were fixed and coated, and their shoot apexes were observed with a scanning electron microscope.
in col seedlings, leaves  <dig> and  <dig>  with trichomes, were clearly visible; primordia for leaves  <dig>   <dig> and  <dig> were visible under higher magnification.
1a-/- 1b-/- shoot apexes showed minimal sam activity: leaves  <dig> and  <dig> were produced, but were smaller than wild type and did not separate to reveal any further leaf primordia .
in order to determine whether a plant hormone or signaling molecule could restore post-embryonic growth to these arrested seedlings, progeny of 1a-/- 1b+/- individuals were germinated on plates containing a variety of plant hormones and signaling molecules and scored for their total seedling lengths at  <dig> days post germination.
signaling molecules tested included the gibberellin ga <dig> , abscisic acid , the auxin  <dig> -d , the ethylene precursor acc , sucrose  and glucose .
we also attempted dark treatment.
none of these treatments restored wild-type sam growth to the quarter of the progeny showing seedling arrest.
1a-/- 1b-/- seedlings did show a significant response to sucrose .
addition of 1% sucrose, which is a growth signaling factor as well as a carbon source  <cit> , to the growth medium promoted growth in col, 1a-/-, 1b-/- and 1a-/- 1b-/-.
in sibling seedlings, the addition of sucrose elicited a twofold increase in seedling length; in the double mutants the increase was five-fold.
germination on 6% sucrose, which signals growth arrest in col plants  <cit> , yielded sibling plants 1– <dig> × the size of seedlings grown on 1/ <dig> murashige & skoog  salts with no sucrose added.
1a-/- 1b-/- seedlings on 6% sucrose were  <dig> × the length of those grown on 1/ <dig> ms salts.
a similar result was observed among 1a-/- 1b+/- progeny germinated in the dark on 1/ <dig> ms salts plates, and the length of 1a-/- 1b-/- seedlings was increased more than 3× under these conditions.
the absolute length of the double mutant seedlings was in all cases still substantially smaller that that of sibling seedlings for a given treatment, and the increase in length was largely due to hypocotyl elongation or root growth.
minimal sam activity was observed.
discussion
axillary branch growth in arabidopsis and other vascular plants is governed indirectly by auxin produced at the primary shoot apex, which acts through an undetermined secondary messenger to repress axillary meristem growth  <cit> .
the shoot morphology of 1b-/- plants indicates a defect in primary sam maintenance.
the primary shoot is shorter than wild type and ends in a whorl of sterile flowers.
the number and length of axillary shoots, both cauline and emerging from the basal rosette, is significantly increased.
notably, axillary meristems are quite viable, producing branches as long as or longer than wild type.
the viability of axillary shoot apices points to a failure to maintain primary sam activity, and a subsequent failure to repress axillary meristem activity.
this defect is not shared by the axillary sams, which match or surpass the activity of their wild-type counterparts.
a similar phenomenon is seen in erecta mutants  <cit>  and may be quite common.
the increased branching in atraptor1b mutant plants may result directly from reduced auxin production at the shoot apex or from a change in the auxin/cytokinin ratio.
however, 1b-/- mutants did not show defects in their ability to sense exogenous auxin .
the endogenous atraptor1a locus cannot fully complement the disruption of atraptor1b despite the fact that their predicted proteins are 80% identical, share all conserved raptor motifs and strongly resolve to a single clade in phylogenetic analysis.
in silico analysis suggests that the failure of atraptor1a to mask atraptor1b lesions may be due to lower levels of atraptor1a expression globally rather than limited tissue-specific accumulation of atraptor1a.
we did not assay for the complementation of atraptor1b lesions by transforming atraptor1b-/- plants with an atraptor1a construct.
in contrast, atraptor1b can fully complement the loss of atraptor1a expression; indeed, a single wild-type atraptor1b allele in 1a-/- 1b+/- plants is sufficient for wild-type growth.
our ability to isolate atraptor1b mutant homozygotes conflicts with a recent report  <cit> .
in that report, mutants homozygous for atraptor1b disruptions were lethal at or before the first asymmetric embryonic division, which is even earlier than the lethality of attor mutant homozygotes.
we are uncertain of the reason for the discrepancy in these results.
we are able to recover 1b-/- plants, but deprost et al.
<cit> , working with the same atraptor1b allele , described them as embryo-lethal.
one possible explanation might be a variation in growth conditions, which could lead to a higher degree of stress on developing embryos in the experiments by deprost et al.
the fact that these authors observed some embryo arrest in wild type control lines supports this possibility.
disruption of atraptor1a yielded no significant phenotype in either our growth conditions or those of the previous report  <cit> .
simultaneous disruption of both atraptor loci resulted in growth-arrested seedlings, which undergo normal embryonic organogenesis but are unable to maintain post-embryonic growth from their sams.
atraptor1a-/- atraptor1b-/- double mutants' sams cease activity after the production of a few leaf primordia.
we conclude that atraptor activity is essential for the maintenance of sam activity, but not for sam assembly or for the initiation of sam activity.
our results indicate that atraptor activity is not essential for embryonic organogenesis.
in contrast, attor-/- mutants arrest development early in embryogenesis  <cit> .
the disparity between the atraptor1a-/- 1b-/- and the attor-/-phenotypes indicates that attor activity in embryonic development does not require atraptor.
thus if attor is acting in a complex in embryonic development, this complex does not require atraptor.
in both yeast and mammalian cells, tor functions in two distinct complexes .
the first of these, torc <dig>  involves raptor and regulates cell growth and translation in response to nutrients  <cit> .
the second of these complexes, torc <dig>  regulates cytoskeletal organization and does not involve raptor  <cit> .
atraptor-independent attor activity in the plant embryo is consistent with the existence of two attor complexes in arabidopsis: 1) a raptor-independent complex critical for early embryogenesis , and 2) a raptor-dependent complex that is dispensable for embryonic development but which is necessary for post-embryonic, meristem-driven plant growth .
CONCLUSIONS
by identifying the phenotypes resulting from partial and total disruption of atraptor activity, we have generated valuable tools for the study of plant tor signaling.
when viewed in the context of previous work on attor  <cit> , our work provides evidence for raptor-independent tor activity in land plant embryonic development – development that, notably, occurs in an environment made nutritionally and environmentally stable via maternal input to growth.
we propose that in plants as in other eukaryotes there are two  tor complexes, only one of which involves raptor.
we further propose that the plant homologue of torc <dig>  involving tor and raptor, has been co-opted in plants from its ancestral role in nutrient sensing and cell growth to regulate the highly plastic post-embryonic growth driven by the plant shoot apical meristem.

 plant development is remarkably plastic. groups of totipotent cells termed meristems, which are maintained throughout the life of the plant, give rise to all post-embryonic organs from roots and leaves to petals and fruit. this allows plants, unlike metazoans, to change their final body plans dramatically in response to environmental, hormonal and nutritional cues. while much has been learned about the determination of cell fates in the embryo and the apical meristems, less is known about the genes that control the growth that occurs in cells emerging from the plant meristem. tor proteins were originally identified in budding yeast as the targets of rapamycin, a potent antibiotic that disrupts cell growth. in both yeast and metazoans, tor proteins mediate translation in response to nutrients. yeast tor and mammalian mtor also regulate cytoskeletal organization. in both yeast and mammalian cells, tor proteins form a complex, torc with gβl and raptor; their yeast homologues are lst and kog respectively. raptor contains protein-binding domains, and the raptor n-terminus shows similarity to a caspase domain, though catalytic activity has yet to be shown. it has been reported that the strength of the tor-raptor interaction is regulated by nutrients, though there is not yet consensus on this point. raptor functions in torc to recruit substrates for phosphorylation by tor; raptor binds tor substrates s6kinase and eif4e-bp, and is necessary for full tor phosphorylation of these substrates in vitro. the tor-raptor complex is thought to mediate the nutrient-sensitive regulation of cell growth; mutants in yeast lacking a functional complex cease cell growth in a manner that mimics rapamycin treatment. a second tor complex, torc involves gβl, rictor and perhaps other proteins. this complex is unaffected by rapamycin, and is thought to mediate tor cytoskeletal regulation. in arabidopsis the single tor homologue, attor, is critical for plant development. attor insertion homozygote embryos undergo cell division but are unable to gain cell volume or undergo apical-basal differentiation. although attor transcripts accumulate in all tissues assayed, an attor-gus fusion transcript is only translated in meristematic tissue and the immediately adjacent expanding cells. a tor-specific microrna conserved between arabidopsis and rice implicates post-transcriptional regulation in attor expression. rapamycin perturbs the activity of the nutrient-sensitive tor complex in yeast and mammals. arabidopsis, however, is insensitive to rapamycin, precluding this approach to the elucidation of arabidopsis tor signaling. here we report a characterization of atraptor1a and atraptor1b, which encode the two arabidopsis raptor homologues. we found defects in both root and shoot growth in atraptor1b disruption lines, resulting in slow leaf initiation, late flowering and increased branching. we also found that atraptor1a atraptor1b double mutant homozygotes undergo normal seedling development, but exhibit only minimal post-embryonic meristem activity. we contrast these results with those found previously for the attor mutant, and discuss the implications of this work for tor signaling in plants.  characterization of the arabidopsis raptor homologues to search for plant homologues of mammalian raptor, we surveyed the completed arabidopsis genome and found two loci that might encode proteins highly similar to raptor at the north ends of chromosomes and we did not find an expressed sequence tag for the first locus. however, we used the 5'-rapid amplification of cdna ends to establish that the locus is transcribed to an mrna with a 5' end consistent with the predicted open reading frame of atraptor1a. the atraptor1a protein is predicted to be amino acids in length. the second locus was represented by a single est. we used 5' race to confirm that the est contained the full orf of atraptor1b. the atraptor1b locus is transcribed to a kb mrna containing exons, and the protein is predicted to be amino acids in length. predicted atraptor1a and atraptor1b proteins show 80% identity over their entirety. to determine the time of divergence between the two atraptor loci, we searched for raptor homologues in available plant genome data using atraptor1a and atraptor1b as query sequences. full-length raptor loci were discovered in the available rice and alfalfa genome sequences. using atraptor1b and partial cdna sequence as guides, we determined putative protein sequences from these loci. alignment of these sequences with atraptor1a, atraptor1b, mammalian raptor, the budding yeast raptor homologue kog and the fission yeast raptor homologue mip1p showed that there is a striking degree of conservation among all raptor homologues. atraptor1b and the saccharomyces cereviseae raptor homologue kog the most divergent member included in the analysis, show 28% identity throughout their length. all plant raptor homologues encode the raptor n-terminal conserved / caspase motif, heat repeats and wd motifs first identified in fission yeast mip and characteristic of all raptor proteins. from this alignment we generated a phylogeny of the raptor homologues. the predicted atraptor proteins resolve to a single clade with a high degree of confidence, indicating that the duplication of the atraptor loci post-dates the divergence of arabidopsis from medicago, and that the loci are likely to encode functionally redundant proteins. identification of knockout mutants in atraptor1a and atraptor1b to gain insight into the function of the atraptor proteins, we searched for insertion alleles of each locus among the publicly available sequenced t-dna insertion lines. insertion mutant lines salk_ and salk_ with disruptions to the atraptor1a and atraptor1b loci, were obtained from the arabidopsis biological resource center. lines homozygous for each insertion were identified via polymerase chain reaction, and rna from floral buds of insertion homozygotes was used for reverse-transcriptase-pcr to assay for accumulation of wild-type transcripts from the disrupted locus. atraptor1a transcripts could not be detected in 1a -/- buds, but were detected in 1b -/- buds. atraptor1b transcripts were not detected in 1b -/- buds, but were detected in 1a -/- buds. both transcripts were detected in wild-type columbia buds. 1b-/- seedling roots grow slowly as a first step toward characterizing these mutant lines, col, 1a-/- and 1b-/- seedlings were germinated on culture medium. col and 1a-/- seedlings were phenotypically indistinguishable. however, b-/- seedlings showed distinct root growth defects: roots were thick, coiled and densely covered with hairs, and the roots had difficulty penetrating the medium. plate-grown 1b -/- seedlings were repeatedly shorter than col or 1a-/- seedlings. although dark grown 1b-/- seedlings were also shorter than col or 1a-/-, the difference in size was much less. while dark-grown etiolated seedlings grow primarily through the expansion and division of embryonic hypocotyl cells, light-grown seedlings grow primarily through the production of cells from the root apical meristem. thus the difference in size between col, 1a-/- and 1b-/- seedlings results primarily from a reduction in root apical meristem growth rather than a general defect in cell expansion or metabolism. the roots of 1b-/- seedlings grown on 90° inclined plates were morphologically wild type but shorter than col roots. when the plates were rotated to lay flat horizontally, new growth from the root apex of 1b-/- seedlings produced thick, coiled roots, while the previously produced root tissue remained morphologically wild type. only those sections of the root not in contact with the medium were hairy. thus the root growth disturbance was a defect in growth into agar rather than in gravitropism, though in the absence of resistance, roots still grew more slowly than wild type. col and 1b-/- root apical meristems were examined using bright-field microscopy. 1b-/- seedling rams contained all cell types present in col rams but their root morphology was much more blunt; this difference appeared to be localized to the 1b-/- zone of elongation. 1b-/- root tips showed a tendency to shed their root caps, although shedding was sometimes observed in col root tips as well. 1b-/- shoots show developmental and morphological defects to measure developmental defects resulting from atraptor insertions, we grew col, 1a-/- and 1b-/- lines on soil, and scored their rates of leaf initiation, time to floral bud initiation, rate of cauline leaf initiation, time to flowering and number of floral shoot apices. 1b-/- plants were smaller, had a slower rate of leaf initiation, and bolted and flowered later than did col or 1a-/- plants. mature 1b-/- plants were conspicuously bushier than col or 1a-/- homozygotes. the primary shoot apex was shorter than that of col plants and ended prematurely in a terminal inflorescence of infertile flowers. the growth of the plant was then taken over by axillary shoots and by secondary shoots from the basal rosette. to quantify this phenotype, col and 1b-/- lines were grown to maturity in 16-hr days, and shoot architecture for an individual plant was scored at the shattering of the first silique. 1b-/- plants showed reduced shoot height, reduced primary stem length, increased axillary branch number and an increased number of secondary shoots compared to col and 1a-/- plants. 1b-/- branch numbers, though variable, were significantly larger than those of wild type or 1a-/-. student's t-tests of sample pairs assuming equal variance yielded p-values of and for 1b-/- vs. col and 1b-/- vs. 1a-/- axillary branch number, respectively, and and for 1b-/- vs. col and 1b-/- vs. 1a-/- secondary shoot number. axillary branch length did not differ significantly from col values. this phenotype became more pronounced later in plant development and was more conspicuous under short days. complementation of the atraptor1b-/- phenotype to confirm that the collection of phenotypes observed in the atraptor1b-/- homozygotes was due to the mutant 1b allele, 1b-/- plants were transformed with a construct containing the atraptor1b orf and 5' utr and driven by the atraptor1b promoter. some of these transformants showed wild-type transition to bolting, shoot branching and root growth. we conclude that the 1b-/- phenotypes described above result from homozygosity for the lesion at the atraptor1b locus. atraptor transcripts accumulate in dividing and expanding cell tissues a combination of in situ rna hybridization and in silico expression analysis was used to determine the rna accumulation pattern of atraptor transcripts in arabidopsis. the atraptor1b cdna sequence was used to generate an atraptor probe to assay transcript accumulation in the shoot tips of wild-type plants. since atraptor1a and atraptor1b show 80% identity through the length of their transcripts, this probe likely detected expression of both loci.atraptor transcripts were detected throughout the shoot tip, in all organs of the differentiating floral bud, and deep into the differentiated inflorescence stem. signal intensity faded with the distance from the shoot apex. this accumulation pattern differed from that of actin, which was more prominent in dividing cells of the apex. notably, atraptor accumulation is not restricted to the primary shoot apex. to obtain an estimate of the relative levels of atraptor1a and atraptor1b in the signal seen in fig. 6a, atraptor1a and atraptor1b locus ids were used to query the genevestigator arabidopsis thaliana microarray database and analysis toolbox. atraptor1a and atraptor1b accumulate in all developmental stages. however, atraptor1b accumulation levels are consistently fourfold higher than those of atraptor1a. a second analysis of accumulation by tissue rather than developmental stage produced similar results. we conclude that atraptor1a and atraptor1b show similar expression patterns. 1a-/- 1b-/- double homozygote mutant seedlings show minimal meristem growth the high degree of similarity between atraptor1a and atraptor1b led us to suspect that they may be at least partially functionally redundant. to eliminate all atraptor activity in a single line, 1a-/- and 1b-/- lines were crossed. no 1a-/- 1b-/- plants could be identified among the f of this cross on soil. phenotypically wild-type 1a-/- 1b+/- plants were identified via pcr using the primer pairs described in materials and methods and their progeny were examined on agar plates. of these, seedlings appeared wild type. it was determined by pcr that these seedlings carried a wild-type 1b allele. the remaining seedlings germinated but showed minimal postembryonic shoot meristem growth. it was determined by pcr that these seedlings were genetically 1a-/- 1b-/-. this genotyping confirmed that the seedling arrest mutant phenotype co-segregated with homozygosity for the atraptor1b mutant allele in the atraptor1a mutant background. we concluded that this seedling phenotype corresponded to the 1a-/- 1b-/- double mutant. we examined 1a-/- 1b-/- double mutant seedling roots via bright field microscopy to ascertain the extent of their post-embryonic growth defect. 1a-/- 1b-/- roots were conspicuously narrower than wild type. the columella, quiescent center, vasculature, pericycle, endodermis and cortex are present though all smaller than wild type; the outer layer of cells is difficult to identify. further up the root, root hairs were clearly visible, indicating that the ram had produced some mature root cell types. to gain a better understanding of the double mutant shoot apical meristem defect, col and 1a-/- 1b-/- 7-day seedlings were fixed and coated, and their shoot apexes were observed with a scanning electron microscope. in col seedlings, leaves and with trichomes, were clearly visible; primordia for leaves and were visible under higher magnification. 1a-/- 1b-/- shoot apexes showed minimal sam activity: leaves and were produced, but were smaller than wild type and did not separate to reveal any further leaf primordia. in order to determine whether a plant hormone or signaling molecule could restore post-embryonic growth to these arrested seedlings, progeny of 1a-/- 1b+/- individuals were germinated on plates containing a variety of plant hormones and signaling molecules and scored for their total seedling lengths at days post germination. signaling molecules tested included the gibberellin ga, abscisic acid, the auxin -d, the ethylene precursor acc, sucrose and glucose. we also attempted dark treatment. none of these treatments restored wild-type sam growth to the quarter of the progeny showing seedling arrest. 1a-/- 1b-/- seedlings did show a significant response to sucrose. addition of 1% sucrose, which is a growth signaling factor as well as a carbon source, to the growth medium promoted growth in col, 1a-/-, 1b-/- and 1a-/- 1b-/-. in sibling seedlings, the addition of sucrose elicited a twofold increase in seedling length; in the double mutants the increase was five-fold. germination on 6% sucrose, which signals growth arrest in col plants, yielded sibling plants 1– × the size of seedlings grown on 1/ murashige & skoog salts with no sucrose added. 1a-/- 1b-/- seedlings on 6% sucrose were × the length of those grown on 1/ ms salts. a similar result was observed among 1a-/- 1b+/- progeny germinated in the dark on 1/ ms salts plates, and the length of 1a-/- 1b-/- seedlings was increased more than 3× under these conditions. the absolute length of the double mutant seedlings was in all cases still substantially smaller that that of sibling seedlings for a given treatment, and the increase in length was largely due to hypocotyl elongation or root growth. minimal sam activity was observed. discussion axillary branch growth in arabidopsis and other vascular plants is governed indirectly by auxin produced at the primary shoot apex, which acts through an undetermined secondary messenger to repress axillary meristem growth. the shoot morphology of 1b-/- plants indicates a defect in primary sam maintenance. the primary shoot is shorter than wild type and ends in a whorl of sterile flowers. the number and length of axillary shoots, both cauline and emerging from the basal rosette, is significantly increased. notably, axillary meristems are quite viable, producing branches as long as or longer than wild type. the viability of axillary shoot apices points to a failure to maintain primary sam activity, and a subsequent failure to repress axillary meristem activity. this defect is not shared by the axillary sams, which match or surpass the activity of their wild-type counterparts. a similar phenomenon is seen in erecta mutants and may be quite common. the increased branching in atraptor1b mutant plants may result directly from reduced auxin production at the shoot apex or from a change in the auxin/cytokinin ratio. however, 1b-/- mutants did not show defects in their ability to sense exogenous auxin. the endogenous atraptor1a locus cannot fully complement the disruption of atraptor1b despite the fact that their predicted proteins are 80% identical, share all conserved raptor motifs and strongly resolve to a single clade in phylogenetic analysis. in silico analysis suggests that the failure of atraptor1a to mask atraptor1b lesions may be due to lower levels of atraptor1a expression globally rather than limited tissue-specific accumulation of atraptor1a. we did not assay for the complementation of atraptor1b lesions by transforming atraptor1b-/- plants with an atraptor1a construct. in contrast, atraptor1b can fully complement the loss of atraptor1a expression; indeed, a single wild-type atraptor1b allele in 1a-/- 1b+/- plants is sufficient for wild-type growth. our ability to isolate atraptor1b mutant homozygotes conflicts with a recent report. in that report, mutants homozygous for atraptor1b disruptions were lethal at or before the first asymmetric embryonic division, which is even earlier than the lethality of attor mutant homozygotes. we are uncertain of the reason for the discrepancy in these results. we are able to recover 1b-/- plants, but deprost et al., working with the same atraptor1b allele, described them as embryo-lethal. one possible explanation might be a variation in growth conditions, which could lead to a higher degree of stress on developing embryos in the experiments by deprost et al. the fact that these authors observed some embryo arrest in wild type control lines supports this possibility. disruption of atraptor1a yielded no significant phenotype in either our growth conditions or those of the previous report. simultaneous disruption of both atraptor loci resulted in growth-arrested seedlings, which undergo normal embryonic organogenesis but are unable to maintain post-embryonic growth from their sams. atraptor1a-/- atraptor1b-/- double mutants' sams cease activity after the production of a few leaf primordia. we conclude that atraptor activity is essential for the maintenance of sam activity, but not for sam assembly or for the initiation of sam activity. our results indicate that atraptor activity is not essential for embryonic organogenesis. in contrast, attor-/- mutants arrest development early in embryogenesis. the disparity between the atraptor1a-/- 1b-/- and the attor-/-phenotypes indicates that attor activity in embryonic development does not require atraptor. thus if attor is acting in a complex in embryonic development, this complex does not require atraptor. in both yeast and mammalian cells, tor functions in two distinct complexes. the first of these, torc involves raptor and regulates cell growth and translation in response to nutrients. the second of these complexes, torc regulates cytoskeletal organization and does not involve raptor. atraptor-independent attor activity in the plant embryo is consistent with the existence of two attor complexes in arabidopsis: 1) a raptor-independent complex critical for early embryogenesis, and 2) a raptor-dependent complex that is dispensable for embryonic development but which is necessary for post-embryonic, meristem-driven plant growth.  by identifying the phenotypes resulting from partial and total disruption of atraptor activity, we have generated valuable tools for the study of plant tor signaling. when viewed in the context of previous work on attor, our work provides evidence for raptor-independent tor activity in land plant embryonic development – development that, notably, occurs in an environment made nutritionally and environmentally stable via maternal input to growth. we propose that in plants as in other eukaryotes there are two tor complexes, only one of which involves raptor. we further propose that the plant homologue of torc involving tor and raptor, has been co-opted in plants from its ancestral role in nutrient sensing and cell growth to regulate the highly plastic post-embryonic growth driven by the plant shoot apical meristem. 
disruption of atraptor1b yields plants with a wide range of developmental defects: roots are thick and grow slowly, leaf initiation and bolting are delayed and the shoot inflorescence shows reduced apical dominance. flowering plant development is wholly reliant on growth from meristems, which contain totipotent cells that give rise to all post-embryonic organs in the plant. to identify genes that regulate meristem-based growth, we considered homologues of raptor proteins, which regulate cell growth in response to nutrients in yeast and metazoans as part of a signaling complex with the target of rapamycin kinase. the data implicate the tor signaling pathway, a major regulator of cell growth in yeast and metazoans, in the maintenance of growth from the shoot apical meristem in plants. these results provide insights into the ways in which tor/raptor signaling has been adapted to regulate plant growth and development, and indicate that in plants, as in other eukaryotes, there is some raptor-independent tor activity. atraptor transcripts accumulate in dividing and expanding cells and tissues.
 flowering plant development is wholly reliant on growth from meristems, which contain totipotent cells that give rise to all post-embryonic organs in the plant. plants are uniquely able to alter their development throughout their lifespan through the generation of new organs in response to external signals. to identify genes that regulate meristem-based growth, we considered homologues of raptor proteins, which regulate cell growth in response to nutrients in yeast and metazoans as part of a signaling complex with the target of rapamycin kinase.  we identified atraptor1a and atraptor1b, two loci predicted to encode raptor proteins in arabidopsis. disruption of atraptor1b yields plants with a wide range of developmental defects: roots are thick and grow slowly, leaf initiation and bolting are delayed and the shoot inflorescence shows reduced apical dominance. atraptor1a atraptor1b double mutants show normal embryonic development but are unable to maintain post-embryonic meristem-driven growth. atraptor transcripts accumulate in dividing and expanding cells and tissues.  the data implicate the tor signaling pathway, a major regulator of cell growth in yeast and metazoans, in the maintenance of growth from the shoot apical meristem in plants. these results provide insights into the ways in which tor/raptor signaling has been adapted to regulate plant growth and development, and indicate that in plants, as in other eukaryotes, there is some raptor-independent tor activity. 