Patent Application: US-31117403-A

Abstract:
the invention relates to the field of plant growth and development , more in particular to the communication between plant cells influencing architectural or phenotypical characteristics such as their rate and pattern of division , orientation of elongation , organogenesis or differentiation patterns . the invention provides a method for modulating plant phenotype or architecture , such as by modulating or changing plant growth , it &# 39 ; s development or defense responses , by modulating its rate or pattern of cell division , orientation of elongation , organogenesis or differentiation patterns , comprising providing a plant or plant material with recombinant ligand - like protein or a functional fragment thereof , said protein or fragment at least comprising an llp box as provided by the invention comprising an amino acid motif xrxxxxgxxxxhx .

Description:
isolated microspores of brassica napus cv . topas at a stage around the first pollen mitosis were cultured either at 32 ° c . or at 18 ° c . the higher culture temperature leads to the formation of embryos and the lower culture temperature leads to pollen maturation ( fig1 ). samples were collected at various days after initiation of the cultures and total rna was prepared according to the procedure described in materials and methods . mrna differential display rt - pcr ( ddrt - pcr , liang and pardee , 1992 ) was used to isolate cdna clones which appeared specifically under embryogenic conditions ( 32 ° c .). the ddrt - pcr gel of fig2 shows a pcr fragment ( named bnllp1 , indicated by an arrow ) that was found in the samples of microspores cultured for 10 days ( globular to heart shaped embryos ) and 16 days ( heart to torpedo stage embryos ) at 32 ° c . ( embryogenic development ), but not in samples of freshly isolated microspores ( t = 0 ), microspores cultured at 18 ° c . ( gametophytic development ) or in leaf . this bnllp1 pcr fragment was isolated from the gel and sequenced after re - amplification and cloning . comparison with dna sequences in ncbi genbank revealed no significant sequence homology with known genes . a cdna library prepared from globular to heart staged embryos was screened in order to clone the full length cdna of bnllp1 . this has led to the identification of a full length bnllp1 cdna ( otherwise known as dd3 - 12 ), as shown in seq id no . 1 ( fig3 ). analysis of this putative protein using signalp programs ( http :// www . cbs . dtu . dk / services / signalp /) indicated that this protein has a 23 amino acid hydrophobic transit peptide . such a signal peptide will be removed during the transfer from inside the cell to outside . it is therefore expected that the final product of this peptide has only 51 amino acids . proteins with such characteristics are normally working as a ligand protein interacting with one or several receptor kinases in the membrane of surrounding cells for signal transduction between cells ( jennifer and meyerowitz , 1999 ). the expression pattern of bnllp1 as determined by northern blot analysis is shown in fig4 . a high level of transcript was found in microspore embryos of a 10 days 32 ° c . culture ( globular to heart shaped embryos ). no signal was detected in root and leaf tissue , but a faint signal appeared in a mixture of flower buds of various developmental stages fig4 ). separate sampling of rna from younger buds ( 1 - 5 mm ), older buds ( 5 - 8 mm ) and open flowers revealed that the highest level of bnllp1 transcript can be found in the youngest flower buds . within a flower a clear signal was found in pistels , but not in anthers and petals . a bnllp1 promoter :: gus fusion was constructed and transferred to arabidopsis using a “ floral dip ” method ( ref ) to determine the expression pattern of bnllp1 in a close relative of brassica — arabidopsis thaliana . transgenic seedlings were selected on plates containing kanamycin ( fig6 ). the gus signal was first detected in the upper part of the embryos at later globular stage . at the heart - shape stage the gus expression is restricted to the top but slightly close to the abaxial side of the cotyledons . further development of the embryo led to the change of the expression of bnllp1 to a narrow tier of cells at the edge of the cotyledon ( see fig5 ). at the cotyledon stage the bnllp1 expression was localized to a ring - shaped region at the base of each cotyledon , but not in the embryo including the root and apical meristems . interestingly during seed maturation , the expression of the bnllp1 was seen in the remnant of the endosperm , a single layer of cells located between the testa and the embryo . the gus expression was continued till the first few days of seed germination . during post - embryonic development , the expression of bnllp1 is restricted to axillary buds , flower buds and mature roots , not in leaf , flower , or vegetative meristems . in arabidopsis each axillary bud will normally form one new inflorescence which has 2 - 5 cauline leaves and indefinite number of flowers . as soon as flower starts to form , no cauline leaf will be produced . generally , only one inflorescence is produced from each axillary bud . in the axillary buds , the expression is restricted to leaf primordia and moved quickly to the abaxial side of the peteols when leafs are expanding ( fig8 c ). in the flower buds , the bnllp1 expression was first seen in the stage 3 flower buds at a periphery of the flower primordia indicating the positions where sepals are forming . in a stage 5 flower , the bnllp1 is no more expressed in the sepals which has already formed , it is in a region between sepal and carpel primordia , where petals and stamens are going to be formed . in a stage 7 flower , when stamens are forming the bnllp1 expression is seen only at the top of the carpel where stigma is forming . the expression of bnllp1 was switched off completely before the flower opens . in roots , the expression of bnllp1 started after root hairs are formed , 6 - 7 days after germination ( fig7 ). no expression can be seen in the hypocotyl and the expression margin between hypocotyl and root are very sharp . within the root , bnllp1 expression was excluded from the epidermal layer on which the root hairs will be formed . the bnllp1 expression was gradually switched off in the cortex and the ground tissue to the vascular boundles , and later to the pericycle and then off completely when the root hair starts to degenerate . apparently the bnllp1 expression is associated with mature roots with well developed root hair . this is the region where root functions dominantly for nutrient intake from soil . no bnllp1 expression was seen during lateral root induction , nor the old root which functions as a supporting and transporting organ . doubled enhanced 35s promoter was used to drive the over - expression of full length of bnllp1 gene ( otherwise known as dd3 - 12 ) in arabidopsis . the transformation was carried out using the floral dip method mentioned previously . three independent transformants with almost identical phenotype were obtained from 2 transformation experiments . these plants are slow growing and late flowering , bolting only 45 - 50 days after seeds were planted instead of 20 days in the wt . a dramatic phenotype of these bnllp1 over - expression plants is their changes in branching patterns ( fig9 ). instead of one branch was formed at each axillary buds , these plants normally have 2 , 3 , 4 , 5 , and even 7 inflorescence produced at the axillary position of cauline leaves . the formation of new inflorescence are gradual , starts with one branch and new ones are formed during the growth of the plants . these bnllp1 over - expression plants are male sterile , no viable pollen can be produced in the flower . the anther also stays very small , in a triangle shape . another change in the bnllp1 over - expression plants is the formation of pin - shaped carpel in 80 % of the flowers ( fig1 ). these pin - shaped carpels are slender structure without formation of ovules inside . a stigma - like structure can be observed at the top of the carpel indication that the expression of bnllp1 may function as a signal cue for ovule induction , rather than the formation of the stigmatic tissue . those 20 % flowers with normal pistil are fertile if pollinated with pollen from wildtype plants . a careful cytological analysis has showed that the bnllp1 ever - expression plants have defects in building up vascular strands , especially in flowers ( fig1 ). general blast or blastp searches through ncbi and arabidopsis database using either bnllp1 cdna sequence or protein sequence showed no significant homology with any known cdna or protein sequences . our first attempt in searching existing protein databases swissprot using blastp in ncbi and the tair ( an arabidopsis database ) genbanks have showed no significant matching sequences . however , based on the sequence alignment between bnllp1 and llp1at proteins we found that the c - terminals of these two proteins are highly conserved , which might be associated with the important function of these genes ( fig1 & amp ; 13 ). we then used the c - terminal sequence to do the database search with a modification of several parameters ( fig1 ). instead of using protein - protein homologous search , we used the c - terminal peptide sequences to search all nucleotide sequences in ncbi and tair databases with 6 - frame translation . this will allow us to access all the possible sequences in the databases . such a search has led us to the identification of a group of proteins with highly conserved c - terminal boxes , thirteen of them are from arabidopsis ( fig1 ), one from cotton and one from soyabean . this is a boxmotif which has never been identified before . we termed it as llp boxmotif . interestingly , all these proteins are very small , ranging from 60 to 120 amino acids ( see for examples fig1 - 22 ). additional members have been identified which belong to the llp family using this search criteria ( fig2 ). all of these proteins with a llp boxmotif have an n - terminal signal peptide with 15 to 32 amino acids , as indicated by signalp analysis ( ttp :// www . cbs . dtu . dk / services / signalp /). such signal peptides control the entry of virtually all proteins to the secretory pathway to outside of the cells . the signal peptide will be cleaved off while the protein is translocated through the membrane . the common features of these signal peptides are a positively charged n - region , followed by a hydrophobic h - region and a neutral but polar c - region . a (− 3 ,− 1 ) rule states that the residue at the position − 3 and − 1 ( relative to the cleavage site ) must be small and neutral for cleavage to occur correctly ( nielsen et al , 1997 ). based on these three common features , it becomes apparent that llp is a new class of protein . they may function as ligands to interact with receptor kinases in the neighboring cells for cell - cell communication . since llp genes encode ligands that are able to interact with membrane bound receptor kinases in order to induce a signal transduction cascade , it is possible to make use of this interaction for other purposes . redesigning a llp ligand in such a way that a stable non - productive interaction occurs between it and its receptor will result in a competition between the modified and the wild - type ligand for receptor binding . substituting certain amino acids in the receptor interaction domain will create a stable , non - functional ligand that will occupy the receptor binding sites , resulting in a dominant negative mutant phenotype where the signal transduction cascade is blocked . this can be used to alter plant architecture . the interacting domains of the ligand and receptor can also be used in a different context , by linking them to other proteins that normally would not interact . in this way new protein - protein interactions can be created in planta . several known proteins have certain similarities to the llp proteins . these are clavata3 protein from arabidopsis and esr protein from maize . they are also small proteins with a signal peptide at their n - terminals . these proteins showed certain similarities with llp in the llp boxmotif as well , certainly the similarity is lower . the most distinct differences are the location of the llp boxmotif . a somewhat alike box in clavata3 and esr proteins is located much further away from the c - terminal end than llp . as mentioned above , most llp proteins shown here have the llp boxmotif 0 - 3 amino acids away from their c - terminal ends . one animal protein , a putative rho / rac guanine nucleotide exchange factor ( rho / rac gef ) isolated from mouse ( accession no . p52734 ), also showed certain homology with llp proteins in the llp boxmotif . in this case , however , the assumed llp boxmotif is much further away from the c - terminal and even closer to the n - terminal ( pasteris , et al , 1995 ). this is the first time that the llp sequence motif has been identified in any organism . in plants we find this motif generally associated with small extracellular ligand - like proteins . given the conserved nature of the llp motifbox , the public sequence databases were searched for sequences or putative orfs that encode proteins containing a k r x ( v / i ) ( p / h ) ( s / t ) g ( p / s ) ( n / d ) ( p / h ) ( l / i ) ( h / n ) domain . many arabidopsis llp box - containing orfs were identified . many of these were not yet annotated in the database . extrapolating from the llp box , the start and stop codons of the orf were identified . the size of the predicted proteins encoded by these orfs ranged from 100 to 250 amino acids , and all had a high probability of encoding a signal peptide at their n terminus . the following arabidopsis orfs belong to the llp family based on the size of the predicted protein , the likelyhood of a amino terminal signal peptide , and the presence of the llp box : located on chromosome 1 , on bag clone f23o10 accession ac018364 ( 34983 until 34660 ) from arabidopsis thaliana located on chromosome 1 , on bac clone f2op5 accession ac002062 ( 108432 until 108788 ) from arabidopsis thaliana located on chromosome 1 , on bag clone t1k7 accession ac013427 ( 8397 until 8769 ) from arabidopsis thaliana located on chromosome 2 , on bag clone f2i9 ( section 4 of 255 of chromosome 2 ) accession ac006069 ( 55097 until 55786 ) from arabidopsis thaliana located on chromosome 1 , on bag clone t7a14 accession ac005322 ( 16956 until 17207 ) from arabidopsis thaliana located on chromosome 1 , on bag clone f12a21 accession ac008113 ( 31668 until 31928 ) from arabidopsis thaliana located on chromosome 1 , on bac clone f15h11 accession ac008148 ( 45836 until 46069 ) from arabidopsis thaliana located on chromosome 1 , on bag clone f27j15 accession ac016041 ( 90633 until 90932 ) from arabidopsis thaliana located on chromosome 1 , on bac clone f9n12 accession ac022355 ( 48056 until 48298 ) from arabidopsis thaliana located on chromosome 1 , on bac clone f2p9 accession ac016662 ( 57033 until 57356 ) from arabidopsis thaliana additionally , a number of expressed sequence tags ( est &# 39 ; s ) and genes with previously unknown functions that were found in the database , belong to the llp family , based on the criteria mentioned above . these include : thaliana , columbia col - 0 , rosette - 2 arabidopsis thaliana cdna clone 701546165 , mrna sequence gi | 58454631 | gb | ai998558 . 1 | ai998558 [ 5845463 ] thaliana , ohio state clone set arabidopsis thaliana cdna clone 701496429 , mrna sequence gi | 5840376 | gb | ai993471 . 1 | ai993471 [ 5840376 ] [ 0060 ] zea mays endosperm cdna library from schmidt lab cdna , mrna sequence gi | 4887284 | gb | ai677383 . 1 | ai677383 [ 4887284 ] rice cdna from immature leaf including apical meristem oryza sativa cdna clone e51222 — 2z , mrna sequence gi | 3763791 | dbj | au030543 . 1 | au030543 [ 3763791 ] soybean glycine max cdna clone genome systems clone id : gm - c1016 - 2901 5 ′, mrna sequence gi | 6094825 | gb | aw119439 . 1 | aw119439 [ 6094825 ] the criteria used to identify these llp proteins can be used to recognise new members of the llp family as they appear in public databases . isolation of differentially expressed genes from the b . napus microspore embryogenesis system as said , microspores of b . napus isolated at the stage around the first pollen mitosis were cultured in vitro at either 32 ° c . or at 18 ° c . ( custers et al ., 1994 ). the higher temperature leads to a high frequency of embryo formation ( sporophytic development ) and the lower temperature leads to pollen maturation ( gametophytic development , fig1 ). samples were collected at various time points ( 8 hr , 10 and 16 days ) after initiation of the culture and analyzed for changes in gene expression using dd - pcr analysis . these time points were selected as being the minimum embryo induction stage ( 8 hr ), the pattern formation stage ( transition from globular to the heart shape , 10 days ) and the differentiation stage ( torpedo embryos , 16 days ). to avoid the occurrence of non - embryogenesis but heat - shock related genes , microspores treated at 41 ° c . for 45 min were used as an additional control . under such condition no embryogenesis was observed . more than 100 bands that showed increased or decreased expression in embryogenic culture were excised from dd - pcr gels , amplified by pcr and used as probes on northern and reverse northern analysis . amplified fragments showing an expression pattern consistent with the original dd - pcr expression pattern was selected for further analyses . sequence information was obtained from 82 bands and used to query publicly available sequence databases . here we present a further characterization of one of these isolated genes , llp1 . identification of llp1 , a gene encoding a small protein with signal peptide the llp1 cdna fragments was expressed in microspore - derived embryos of b . napus 10 days after the 32 ° c . induction treatment ( globular to heart - shaped stage ), but not in freshly isolated microspores ( t = 0 ), nor in microspores cultured at 18 ° c . or 41 ° c . or in leaves ( fig2 indicated by an arrow ). this 368 bp dd - pcr fragment was sequenced after re - amplification and cloning ( fig3 bottom strand ). to obtain a full - length llp1 cdna , we screened a cdna library prepared from globular to heart - shape microspore - derived embryos using the llp1 dd - pcr fragment as a probe . a 417 bp cdna fig3 topstrand ) with a single open reading frame ( orf ) was identified . this cdna encodes a predicted 8 . 3 kda peptide with 74 amino acids ( fig3 , underlined ). analysis of the llp1 protein using signalp program ( http :// www . cbs . dtu . dk / services / signalp /) indicated that , with 99 . 6 % probability , llp1 has a 23 - amino acid hydrophobic transit signal peptide at its n - terminus ( fig3 a , sequence before the . signal peptides control the entry of proteins to the secretion pathway ( nielsen et al ., 1997 ) and are cleaved off during the transfer from the cytoplasm to the outside of the cell . cleavage of the llp1 signal peptide would produce a mature protein of only 51 amino acids . queries using the llp1 sequence to protein and expressed sequence tag ( est ) databases revealed no significant similarity with known proteins or cdnas . however , comparison of llp1 with dna sequences in the genbank database revealed homology with a recently sequenced arabidopsis p1 genomic clone ( muj8 ) located on chromosome 3 ( 37 cm on physical map ). this region of the genomic dna in arabidopsis has one orf with three candidate start codons . structural comparisons of the arabidopsis orf with the b . napus llp1 gene suggests that the second start codon is functional in this sequence , resulting in a peptide with the same length as the llp1 protein ( fig1 , atllp1 . pro ). we named the arabidopsis orthologue as atllp1 . both llp1 and atllp1 lack introns . interestingly , no match to the atllp1 gene was found among 114 , 351 ests available in arabidopsis database , although rt - pcr showed clearly the existence of the transcript ( data not shown ). llp1 is readily detectable by northern analysis ( see below ) and is therefore not likely to be under represented due to it &# 39 ; s abundance . a more likely explanation for the under representation of llp1 ests could be that most cdna libraries are constructed using fractionated cdna , therefore genes like atllp1 with short transcripts may present in these libraries in very low abundance . furthermore , due to its small orf , the atllp1 gene has not been annotated as encoding a gene by the arabidopsis genome - sequencing project . this could be a common problem for small unknown proteins . signalp analysis showed that atllp1 has 99 . 8 % probability of carrying a 24amino acid signal peptide at its n - terminal . over the 225 bp coding region , these two peptides shared 76 . 4 % and 68 % sequence identity at the dna and protein level respectively . southern blotting ( data not shown ) and database searching in the complete arabidopsis genome sequence showed that atllp1 is a single copy gene located at the 37 cm position on chromosome 3 . the map position is consistent with our data obtained from the analysis of recombinant inbred lines , which was carried out before this part of the genome was sequenced ( data not shown ). protein sequence alignment between llp1 and atllp1 showed that the longest stretch of conserved amino acids was present at the c - terminus ( fig1 ). we then used a 31 - amino acid c - terminal peptide sequence to query public databases and found 18 other similar genes in arabidopsis genome . additionally , we also found some matching ests from arabidopsis , tomato , soybean , medicago and cotton and some genomic sequences with similar orfs ( fig1 and 13 ). alignment of these proteins revealed a conserved motif , krxxpxgpxplh , was present in all four proteins ( fig1 and 13 ). this motif has not been previously described . we termed it the llp box . among these related sequences two genes , clv3 from arabidopsis and the zmesr from maize , have been studied before although no linkage between these two genes was observed earlier . the trip box provided here allows us to identify the new gene family . clv3 is the first protein ligand identified from higher plants , and interacts with the clv1 / clv2 receptor kinase complex to mediate signal transduction within shoot apical meristems ( fletcher et al ,, 1999 ). the zmesr protein is encoded by a gene expressed in a restricted region of endosperm around the embryo ( opsahl - ferstad et al ,, 1997 ). outside of the llp box , the llp1 proteins showed weak similarity with clv3 ( fig3 b , indicated in bold ), but not with zmesr . in the llp1 and the atllp1 proteins , llp box is located two amino acids before the c - terminal whereas in zmesr the llp box is located 43 - aa before its c - terminal end . clv3 has an additional 16 - amino acids after the llp box ( fig1 , 13 ). as said in fletcher et al , the clv3 gene encodes a protein of 96 amino acids that was thought to show no appreciable similarity to other sequences or sequence motifs of known functional domains , consequently , gaining the insight of a group of proteins sharing a common feature , namely the llp box , and a common action mechanism ( binding to a receptor and eliciting a phenotypic response ) is provided herein for the first time . the cloning of clv3 thus allowed fletcher a view of meristems as collections of intercommunicating cells , each sythesizing and secreting its own set of protein ligands and responding to its neighbors through action of its own complement of transmembrane receptor kinases , however , even though it is well - understood that other protein ligands must exist in many proteins ( inside or outside the meristem , for that matter ), fletcher et al provide no method for finding or identification of such ligand . similarly , in opsahl - ferstad et al . a number of maize genes were identified with a specific expression pattern , signal sequence and size . conserved domains found among these genes do not include the llp box , this is located in what they define as the variable region . functions of esr as proposed by the author include physical separation of embryo and endosperm ( a structural role in the cell wall ), or nutrition of the embryo ( to be taken up and consumed ). no mention was made of a possible function in signal transduction as a ligand to direct differentiation of either embryo or endosperm . llp1 is expressed in a defined small number of cells during embryonic and post - embryonic development the dd - pcr experiment showed the expression of llp1 in microspore - derived embryos , but not in microspores / pollen and leaf tissue ( fig2 ). northern blotting was used then to further characterize the llp1 expression pattern in additional tissues . northern blot analysis showed relatively high amounts of llp1 mrna in the globular to heart - shape staged embryos and in young flower buds ( 1 - 5 mm in size ), lower levels in older flower buds ( 5 - 8 mm ) containing binucletae to trinucleate pollen , and almost undetectable levels in open flowers at the anthesis stage ( fig4 ). within flower buds , expression was detected in pistils , but not in anthers and petals ( fig4 ). no detectable signal was observed in leaves . to study llp1 expression in more detail , a 1 , 060 bp genomic sequence ( genbank accession number af343658 , from 0 to 1 , 060 bp ) located up - stream of the llp1 start codon was isolated from b . napus by genome walking , fused to the e . coli β - glucuronidase a ( gus ) reporter gene and transformed to arabidopsis . we analyzed llp1 expression during embryonic and post - embryonic development in several transgenic arabidopsis lines . the result was consistent with the northern blot analysis , and among different transgenic lines . to define the precise expression pattern of llp1 during embryogenesis , zygotic embryos from transgenic plants were excised from seed and then stained for gus activity . hoyer clearing procedure caused the diffusion of the gus staining , therefore the results are also presented diagrammatically based on observation under a dissection microscope . as shown in fig5 a , llp1 expression was first detected in the upper region of the late globular embryos . at the heart - shape stage , the gus staining was restricted to a few cells at the top and at the abaxial side of the cotyledon primordia ( fig5 a and b ). further development of the embryo led to a change of the llp1 expression to a narrow tier of cells at the edge of the cotyledon in torpedo - shaped embryos ( fig5 a ). at the bent cotyledon stage llp1 expression was localized to a ring - shaped region at the base of each cotyledon , but was absent from the shoot meristem itself ( fig5 a and d ). during seed germination , llp1 expression was observed in the aleurone , a single layer of endosperm located between the testa and the embryo ( fig5 e and f ). freshly germinated seedlings showed no gus expression . the first detectable gus signal was seen in the root hair region when the main root was longer than 1 cm ( 5 days after plating ). a sharp difference in gus staining was seen between the root , which was stained very strong , and the hypocotyl that was always negative for gus activity ( fig6 b ). within the root , llp1 expression was excluded from the epidermis layer and , no gus staining was seen in the root hairs ( fig6 b and c ). occasionally , llp1 expression was observed in the quiescent center of the primary root ( fig6 d ). we are not sure if it is from the promoter activity or just background staining sinc 1 ) it is not consistent ; 2 ) other researchers have observed such background activity before . along the long axis of the root , llp1 expression was seen only in the well - developed root hair region along a total length of 1 cm or less ( fig7 ). neither the root tip , nor the elongation zone and the secondary thickening zones exhibited any gus staining ( fig6 e and a ). although llp1 expression was observed in all cell layers in freshly germinated primary roots except epidermis ( fig7 d ), at later stages the expression was restricted to the pericycle layer outside the xylem elements ( fig7 b and c ). in radial sections , llp1 expression was observed in two or three pericycle cells facing the protoxylem , whereas the pericycle cells next to the protophloem were always negative ( data not shown ). in arabidopsis , the central cylinder is of the diarch type i . e . with two protoxylem elements and at a right angle to 2 protophloem elements . the pericycle at the outmost layer of the central cylinder is composed of an average of 12 cells in circumference and the lateral root always initiates from the pericycle cells that face to the protoxylem ( dolan et al ., 1993 ). during lateral root formation , we observed that llp1 expression was completely down - regulated in the region , as well as in the cells adjacent to the protoxylem ( fig6 c ). the llp1 expression pattern , together with the different potential in lateral root induction in this layer , indicates that different cells in the pericycle ring may have different developmental potentials in relation to their positions . the expression in lateral roots re - assumed as they matured enough and became covered with root hairs . in summary , llp1 expression in roots is associated with few pericycle cells in the maturation zone . this region of the root is normally covered with root hairs and functions predominantly for nutrient intake from the soil . in above ground tissues llp1 expression was restricted to floral and inflorescence meristems . the first detectable gus signal was seen in the axillary inflorescence ( also called paraclade ) primordia of 8 - day old seedlings carrying 3 - 4 leaves . the primary vegetative meristem did not show any expression before switching to an inflorescence meristem . the determination of the inflorescence meristem may occur earlier in the axillary buds than in the primary vegetative meristems , since all the axillary buds at the time of initiation are determined to form a paraclade ( inflorescence shoot ). in arabidopsis ( c24 ), each axillary bud will give rise to one paraclade with 3 - 5 cauline leaves before the production of an indefinite number of flowers . once the flower starts to form , no additional cauline leaves will be produced . in the young axillary buds , llp1 expression was observed in the periphery of the meristem , at the point where the cauline leaves will emerge ( fig8 ) and appears to be restricted to the l1 layer . this expression pattern continued until the young leaf primordia were formed and was switched off before the expansion of the leaves ( data not shown ). the central inflorescence meristems were always negative in llp1 expression ( data not shown ). in floral meristems , llp1 expression was first observed in stage 2 flower ( smyth et al ., 1990 ) buds at the regions where sepal primordia will be formed ( data not shown ). this expression pattern continued until stage 3 , which marks the sepal primordia , at which point we observed asymmetrical llp1 expression between the medial and . radial sepals . llp1 expression appears to initiate earlier and is stronger in the radial sepal , which also emerges before the medial sepal . such an asymmetrical flower development has not been observed in the morphological analysis carried out in arabidopsis by smyth et al ( 1990 ), but was previously demonstrated in b . napus ( polowich and sawhney , 1986 ). in stage 4 flower buds , llp1 expression was restricted to the grooves between the sepal primordia and the central meristem and disappeared completely in stage 6 flower buds when the petal and stamen start to form ( data not shown ). in stage 7 to 11 floral buds , llp1 expression was only observed at the top of the pistil where the stigma will form . the expression of llp1 in flower buds is switched off shortly before the flower opens . [ 0085 ] fig1 shows the criteria we used to search various databases ; fig1 shows llp proteins identified in arabidopsis genome . from the fully sequenced arabidopsis genome , it is possible to see how many llp genes are present . this has led to the identification of 19 llps . the map position of the llp genes were showed in fig3 . although a few of the llp genes have est sequences available , none of these 19 llp genes have been annotated as a gene by the genome sequencing groups . the distribution of these llp genes seems not random . at the bottom of chromosome 1 , there is a big cluster of llp genes , no llp has been found in chromosome 4 ( fig3 ). it is interesting to notice that except the functional conservation through this group genes , encoding small proteins with n - terminal signal peptide and c - terminal conserved llp box , none of them have redundant copies ( paralogues ) in arabidopsis genome . in another words , none of these llps shares more than 50 % identity at the peptide level . this could be the nature for this group of genes . several reasons can be proposed : 1 ) the secondary structure of these peptides is more important for their functions than the amino acid order . this has been seen in smc proteins which have two rod regions have highly conserved coiled coil structure but flexible in primary sequence . 2 ) the sequence flexibility allows precise interaction with corresponding receptor kinases . 3 ) the critical importance of such proteins requires single copy in a plant genome . a similar search has been carried out in other available databases using the criteria we set up as mentioned above . this has led us to identify llp genes in species such as rice , medicago , tomato , etc . ( fig1 ). all these gene identified showed s structural conservation as those ones from arabidopsis . ectopic expression of llp1 in arabidopsis leads to a consumption of the meristem without affecting the induction of lateral roots and side shoots a double enhanced 35s promoter , which is constitutively expressed in most plant tissue , was used to drive the expression of the b . napus llp1 cdna ( 35s :: llp1 ) in arabidopsis . among twenty - five independent transformants obtained , four lines ( a , b , c and d ) showed similar aberrant phenotypes : slow growth and late flowering . bolting occurred only 40 - 45 days after seeds were planted , instead of 20 days in the wildtype . one line ( line d ) was male and female sterile and gave no seed for further analysis in the next generation . genetic analysis of the remaining three lines indicated that their phenotypes were inherited in a mendelian fashion . plating of single insertion heterozygous lines on kanamycin selection media showed that the phenotype is always associated with the transgene , the ratio is consistent with phenotype segregation in soil without kanamycin . dramatic changes in root development were observed in all four over - expression lines . freshly germinated 35s :: llp1 seedlings showed little difference from the wildtype seedlings . however , root growth in 35s :: llp1 plants was retarded ( fig2 , a to d , 12 - day old ). root hairs formation and the initiation of lateral roots were normal in 35s :: llp1 roots ( fig2 , b and d ), further growth of the lateral stopped shortly after root hair formation . consequently , 35s :: llp1 over - expression led to the formation of seedlings with short roots fifteen days after seed germination , the transgenic plants had produced 4 to 8 short roots , with an average length of less than 1 cm , while in the same period of time the main roots in wildtype seedlings reached a length of more than 10 cm , with several side roots of different lengths . root hairs in the over - expression lines were formed almost to the tip of the roots ( fig2 d ). 35s :: llp1 root tips also appeared to be narrow and pointed , as compared to the wildtype roots . root geotropism was not affected in 35s :: llp1 seedlings ( data not shown ). tissue clearing , followed by nomarski microscopy of 35s :: llp1 roots showed that root meristematic tissue was gradually consumed during root growth and development and used to form differentiated cells . as shown in fig1 e and f ( 7 days after plating ), clear differences in the root region could be observed between wildtype and over - expression seedlings . in the wildtype roots , cells were arranged regularly with clear size and shape differences between the root cap , root meristem , elongation zone and maturation zone ( fig2 e ). in llp1 over - expression plants the root meristem zone and the elongation zone became shorter , which was followed immediately by the formation of highly vacuolated cells which were typically seen in the root hair region ( fig2 f ). at this developmental stage the quiescent center was still recognizable . ten days after germination treatment , the root meristem had almost entirely disappeared ( fig2 g ). only a small number of meristematic cells were present in the root tip . these cells were adjacent to highly vacuolated cells that are normally located at the root hair region . the elongation zone and the quiescent center were hardly recognizable ( fig2 g ). the root meristematic cells and the quiescent center had totally disappeared in 2 - week old 35s :: llp1 seedlings ( fig2 h ). all the cells in this region became highly vacuolated and exhibited a thickening of their cell walls . xylem elements reached the central cell region ( fig2 h , indicated by an arrowhead ). abnormalities were also observed in the root cap , although starch grains were still visible ( data not shown ). no evident difference in root structure was seen in wildtype root during this growth period ( data not shown ). the same meristem defect observed in primary roots was also observed in the secondary roots . in conclusion , ectopic expression of llp1 under control of the 35s promoter appeared to have no influence on root induction , but it had a strong effect in promoting the differentiation of meristematic cells -- meristem is consumed faster than it can be regenerated . ectopic expression of llp1 led to similar changes in shoot and floral development observed for root development . all four independent transformants showed a short branch phenotype . line a had a weaker phenotype than line b and c , and produced about 1 - 3 paraclades in total with relative high amount of seeds produced . these inflorescences stopped to form new flowers after producing 10 or less siliques each , instead of 30 to 40 each in the wildtype . line b and c were almost completely male sterile , resulting occasionally in a small number of seeds ( less than 30 per plant ) under normal growth condition . these seeds probably originated from cross - pollination , since no viable pollen could be detected in the anthers of line b and c flowers . genetic analysis was difficult to carry out with such a limited number of seeds . about one third of the flowers formed in plants from line b and c plants had pin - shaped pistils that showed no ovule development and consequently no seed formation ( fig1 ). the other two thirds of the flowers had normal pistils that were able to produce seeds if pollinated with wildtype pollen . no pin - shaped pistils were observed in line a , which had a weaker phenotype . northern blot analysis revealed that the level of llp1 mrna in all these four lines was much higher than those transgenic lines with a wild - type phenotype ( data not shown ). the short branch and pin - shaped pistil phenotypes could be the consequence of the consumption of inflorescence and floral meristems , similar to what was observed in root meristems with the llp1 over - expression . additionally , ectopic llp1 expression of seems to stimulate the formation of paraclade from the axillary buds . instead of the single paraclade normally produced from each wildtype axillary bud ( fig9 ), multiple paraclades were commonly formed in the 35s :: llp1 over - expression lines ( b and c ), particularly in the axils of cauline leaves ( fig9 b and c ). up to 7 paraclades were sometimes observed to regenerate from one axil ( data not shown ). these paraclades normally emerged sequentially , rather than simultaneously . the terminal flower 1 mutant also shows an increase in branch formation , however in this mutant , the multiple shoots are formed in the axils of rosette leaves , and only occasionally from cauline axils ( grbic and bleecker , 2000 ). ectopic expression of llp1 leads to defects in the formation of continuous vascular network in flowers aberrant vascular development was also observed in flowers of 85s :: llp1 over - expression lines . in the wildtype flowers , vascular bundles are formed at stage 9 by extension from the main stem up to flower buds ( fig1 a ). xylem elements were established first in sepals and followed by pistils , stamens and petals , resulting in a complete vascular network . in 35s :: llp1 flower buds ( lines b and c ), regional vascular formation without connection to the stems was observed ( fig1 b and c ). the failure to form a vascular connection seems to be associated with the formation of pin - shaped pistils , since this phenomenon was not observed in flowers with normal pistils . however , not all flowers with pin - shaped pistils have discontinuous vascular towards the main stem . some flowers did form continuous xylem connections , although the number of xylem elements was reduced as compared to the wildtype . local xylem formation as vascular islands was also observed in both sepals and petals ( data not shown ). such vascular islands were observed in flowers with normal and pin - shaped pistils . no vascular bundle was formed within these pin - shaped pistils . expression of llp2 gene ( sense strand ) under the control of double enhanced camv 35s promoter llp2 coding region was amplified by pcr and cloned in both sense and anti - sense orientations to be expressed under the control of double enhanced camv 35s promoter ( using the same over - expression vector mentioned above ). transgenic plants were obtained by selection on kanamycin - containing media . one over - expression plant showed defective in reproductive development ( fig2 a ). the plant continues produce leaves . occasionally one of two flower can be formed . ( fig2 b ). detailed observation showed that such flower has normal sepal and petal , but reduced number of stamen and no pistil ( fig2 c ). the inflorescence meristem terminated quickly before further flower formation ( fig2 d ). expression of llp2 anti - sense under the control of double enhanced camv 35s promoter over - expression of llp2 anti - sense under the control of double enhanced 35s promoter leads to plants with soft and short stems . each inflorescence produces 2 - 6 siliques instead of 25 to 35 in the wildtypes . the number of seeds in each silique was also greatly reduced . it is likely that the over - expression of llp2 anti - sense affected the vascular structure of the plants . genetic analysis showed that the phenotype is associated with the t - dna insertion . tissue specific promoter could be used in combination of the llp2 anti - sense gene to modify the vascular structure of other plant species . expression of llp11 gene under the control of double enhanced camv 35s promoter llp1 coding region was amplified by pcr from genomic dna . the gene was expressed under the control of double enhanced camv 35s promoter ( using the same over - expression vector mentioned above ). 78 % t0 plants over - expressing llp11 gene ( sense strand ) showed phenotypes . based on the phenotype differences , the t0 plants can be divided in three classes : light , medium and severe phenotype lines ( fig2 ). the “ light phenotype ” plants can produced a few inflorescence although the primary one often stopped prematurely . the “ medium phenotype ” plants showed greatly reduction on inflorescence formation . normally a very or a few very short inflorescence can be produced , with few siliques . the “ severe phenotype ” plants do not form any inflorescence , therefore , can not be carried to the next generation . several “ light phenotype ” plants were analyzed in the following generations since enough seeds were available . among the lines analyzed , we observed two different phenotypes that are slightly different from the phenotypes we observed in the to generation : low fertility ( fig2 a ) and slow growing and reduced inflorescence formation phenotype ( fig2 b ). in some lines both phenotypes can be observed and in other lines only one phenotype was observed . genetic analysis showed clearly that the low fertility and slow growing phenotype were caused by over - expression of the llp11 gene , since both traits showed to be dominant and linked to the t - dna in segregation . the slow growing phenotype can be seen in both root and shoot development , producing plants with short roots and small leaves . some low fertility lines (# 67 - 6 , fig2 a ) showed no reduction on vegetative growth . the plants have long paraclade with very short siliques ( because of no or a few seeds produced in each silique ). it is possible that the llp11 genes ( sense and anti - sense approaches ) can be used in combination with different promoters to control growth behavior and pollen development . the promoter region of llp12 ( 1 kb before atg ) was cloned in front of the gus reporter gene in a pbinplus vector . transgenic plants were obtained using the flower dip method mentioned above . gus expression analysis was carried out in leaves , stems , axillary buds , flowers and siliques in 30 independent transgenic lines . the results showed , with certain variation in gus staining , that the llp12 was expressed in immature pollen grains and the pedicel region ( the connection between flower and the stem ) of the flowers ( indicated by diagrammatic drawing in fig2 ). gus analysis in root development will be carried out in the near future . expression of llp12 gene under the control of double enhanced camv 35s promoter several transgenic plants expressing llp12 gene showed more or less the same phenotype . the primary shoots were stopped early and multiple side shoots were formed afterward ( fig2 , a and b ). the plants have very thin and short inflorescences , with no ( fig2 b ) or a few seeds ( fig2 a ) produced . the reduced seedset seemed to be caused by male sterility since seeds can be produced when cross - pollinated with wt pollen . flower development was normal . phenotype segregation can be seen clearly in the in the next generation when seeds were planted on germination plates with or without the selection agent ( km ). during the seedling stage , the transgenic plants have smaller rosette leaves and reduced root elongation . the phenotype segregation could also been seen clearly when seeds were sowed directly in soil ( fig2 c ). at later stage of the inflorescence development , the paraclade showed zigzag arrangement ( fig3 , a and b ). instead of new flowers formed from the side of the inflorescence , in this case , the new flowers formed at the terminal position of the paraclade , whereas the inflorescence were produced at the side . the pedicel ( the joint between stem and flower or silique ) was also much shorter ( fig3 b ) than that in the wt plant . the low fertility and short peduncle phenotype seem consistent with the expression pattern of the llp12 gene . the retarded growth of pedicel may be associated with the suppression function generally seen in most llp genes . genetic analysis showed that such phenotypes are dominant traits and linked to the t - dna ( fig3 , wt plants have been removed from the top picture ). the male sterility caused by llp12 over - expression could be used to modify the reproduction behavior or in hybrid seed production . rt - pcr to test if the llp orfs are real genes , and where do they expressed since most of the llp genes were identified from the genome sequence based on the criteria we set up , it is not sure if all of them are real expressed genes . rt - pcr was used to check the expression profile of these orfs . total rnas were isolated from various tissues of arabidopsis and treated with dnase to remove contamination from genomic dnas . rt - pcr was performed using poly ( t ) as a primer . for the positive control and the quantitative measurement , actin8 gene was used as positive control since it is a ubiquitously expressed gene . two primers , one located at the beginning of the orf and one before the stop codon , were used to perform the pcr reaction . when rna was used to do the pcr , no product has been seen , indicating that genomic contamination have been removed . positive control was carried out using genomic dna these experiments revealed for example that llp2 , llp9 , llp12 and llp18 are genes with different expression profiles . llp2 was expressed in all tissues tested . llp9 was only expressed in different stages of flowers , not in roots , leaves , stems , etc . llp12 showed higher expression in different stages of flower , but also in other tissues tested . llp18 showed expression only in roots . two genes , llp5 and llp7 , showed negative in the rt - pcr analysis in the tissues tested . in summary , these rt - pcr experiments showed that most llp genes identified using the criteria we established are genes of which the expression is different from one another . the cultivation of the doubled haploid brassica napus l . cv . topas plants and the isolation of microspore and pollen grains was performed as described . plants were raised all year round in a phytotron room at 18 ° c . with a 16 h photoperiod . microspores and pollen were isolated by disrupting flower buds with a pestle in nln medium ( lichter , 1982 ) containing 13 % ( w / v ) sucrose ( nln13 ). late unicellular microspore and early bicellular pollen were cultured in nln13 medium at a density of 40 , 000 cells / ml , either at 18 ° c . ( gametophytic development ) or at 32 ° c . ( embryogenic development ). total rna from microspore cultured at 18 ° c . ( 8 h ), 32 ° c . ( 8 h ) or 41 ° c . ( 45 min ) was isolated using an extraction buffer containing a 1 : 1 mixture of phenol and 0 . 1 m licl , 10 mm edta , 1 % sds , 0 . 1 m tris - hcl ( ph 8 . 0 ). one ml of hot ( 60 ° c .) extraction buffer was added to the microspore pellet ( approx . 10 6 microspore ) and the homogenate was rigorously vortexed in the presence of glass beads . after centrifugation the aqueous phase was extracted with an equal volume of chloroform and the rna was precipitated at − 20 ° c . by the addition of ⅓ vol of 8 m licl . the pellet was washed with 70 % ethanol dried and dissolved in diethylpyrocarbonate ( depc )- treated water . all other total rna samples were obtained by grinding the plant material in liquid nitrogen with a mortar and pestle , and subsequent extraction of the fine powder using trizol reagent ( gibco - brl ). genomic dna was isolated from leaf tissue according to fulton et al ., 1995 , and digested with the specified restriction enzymes according to procedures suggested by the manufacturer ( gibco - brl ). differential display ( liang and pardee , 1992 ) of mrna was performed using rnamap kit b ( genhunter , usa ) according to the manufacturer &# 39 ; s recommendation . total rna from freshly isolated microspore , microspore cultured at 18 ° c . ( 8 h ), 32 ° c . ( 8 h , 10 d , 16 d ) or 41 ° c . ( 45 min ), and leaf tissue of b . napus was isolated as described above and dnase i treated using the messageclean kit ( genhunter ). differential display was carried out on two independent 8 h cultures of 18 ° c . and 32 ° c . a real heat - shock was given by treatment of microspore at 41 ° c ., a condition that does not lead to embryogenesis in microspore of this developmental stage . dnase - free total rna samples ( 0 . 2 μg ) were used for the first strand cdna synthesis . four t 12 mn anchor primers ( where m is degenerate a , c , g and n is either a , c , g or t ) were used in four reverse transcription ( rt ) reactions . pcr amplification of one - tenth of the first - strand synthesis cdna products was done in the presence of [ α - 33 p ] datp . five decamers ( ap 6 to ap 10 ) were used in combination with the respective t 12 mn . all pcr steps were performed using the perkin - elmer genamp 9600 system and amplitaq polymerase from perkin - elmer . the amplified [ α - 33 p ] datp labeled cdnas were resolved on 6 % denaturating polyacrylamide gels containing 7 m urea . after drying the gels on whatman 3mm paper and autoradiographic detection of bands , differentially expressed cdnas were excised and eluted according to the manufacturer &# 39 ; s instructions . cdnas were then re - amplified using the same pcr conditions and primers as before . pcr products were analysed on a 1 . 2 % agarose gel and cdna fragments of interest were eluted and cloned into the pgem - t vector ( promega ). to confirm the differential display pattern the cloned cdnas were used as probes for rna blot hybridizations . dna and rna gel blot analyses dna fragments were separated in 1 % agarose and transferred overnight onto hybond - n + ( amersham ) by capillary blotting with 20 × ssc . for rna gel blot analysis , 10 μg of total rna was denatured with glyoxal prior to electrophoresis and blotting onto hybond - n + membrane . after ultraviolet cross - linking the membranes were hybridized with a [ 32 p ] random - primer - labeled probe of the dd - clone of bnllp1 . membranes were hybridized overnight at 65 ° c . in 10 % dextran sulphate , 1 % sds , 1 m nacl , 50 mm tris - hcl ( ph 7 . 5 ) and washed first 30 min twice at moderate stringency ( 65 ° c ., 2 × ssc , 1 % sds ), followed by two 30 min high - stringency washes ( 65 ° c ., 0 . 2 × ssc , 0 . 5 % sds ). poly ( a ) + rna was isolated from total rna of globular to heart stage b . napus microspore embryos using poly ( a ) quik columns ( stratagene ). five μg poly ( a ) + rna was used as starting material for the construction of an uni - zap xr cdna library ( stratagene ). approximately 10 6 plaques were screened under high - stringency conditions with the cdna as isolated by ddrt - pcr ( fig3 ). one positive clone was isolated , purified and sequenced ( fig3 ). the universal genome walker kit ( clonetech ) was used to isolate genomic dna fragments lying upstream of the bnllp1 atg start codon . pools of uncloned , adaptor - ligated brassica napus cv topas genomic dna fragments were constructed and used to isolate bnllp1 genomic sequences by nested pcr . the primary pcr made use of the outer adapter primer ( ap1 ) supplied by the manufacturer and a bnllp1 specific primer with the sequence : the nested pcr made use of the nested adapter primer ( ap2 ) supplied by the manufacturer and a bnllp1 specific primer with the sequence : the primary pcr mixture was then diluted 1 : 50 and used as template for nested pcr . both the primary and nested pcrs were performed as recommended by the manufacturer . the nested pcr products were cloned into the pgemt vector ( promega ) and sequenced . pcr products corresponding to the 5 ′ untranslated genomic region of bnllp1 cdna were identified ( fig1 ). the construction of a plasmid vector containing the bnllp1 cdna under control of the double cauliflower mosaic virus 35s promoter with d amv translational enhancer was as follows . the complete coding region of bnllp1 was already cloned into the gst fusion vector pgex4t - 2 ( amersham pharmacia biotech ). this plasmid was cut with the restriction enzymes bamhi and xhoi . the 231 bp bnllp1 fragment was isolated and ligated into the vector pgd121 ( containing a double 35s promotor with amv enhancer and a pbinplus backbone ), already cut with the restriction enzymes bamhi and xhoi . this construct was confirmed by sequencing , and transformed to a . tumefaciëns c 58pmp90 . the promotor bnllp1 - gus was made as follows ; a 1060 bp bnllp1 promotor fragment ( obtained by genome walking ) cloned in pgem - t ( promega ) was used for this construction this construct was used in a pcr with the primers : p312 - 1 5 ′- cgctctagagttctatctttgtcaaaaaaaa - 3 ′ anneals on the promotor , just before the atg . as proofreading polymerase pfu ( stratagene ) was used . pcr protocol : 45 seconds at 94 ° c ., 60 seconds at 40 ° c ., 4 . 5 minutes at 72 ° c . [ cycle repeated twice ] followed by a 45 seconds at 94 ° c ., 60 seconds at 54 ° c ., 4 . 5 minutes at 72 ° c . [ cycles repeated 18 times ] followed by 3 minutes at 72 ° c . and a 4 ° c . hold . the obtained fragment was cut with the restriction enzymes hindiii and xbai , and ligated into the prap2t / gus vector ( containing gus intron and the nos terminator and a puc vector as backbone ) that was already digested with hindiii and xbai . this constuct was digested with paci and asci and a fragment containing the bnllp1 promotor , gus - intron and the nos terminator was isolated and ligated into pbinplus , digested with paci and asci . the obtained vector was confirmed by sequencing and transformed to a . tumefaciëns c 58pmp90 . [ 0121 ] arabidopsis thaliana ecotype c24 was used as the recipient in transformation experiments . plants were transformed using the flower dip method described in clough and bent ( 1998 ). plant materials were glued to a copper stub using conductive carbon glue and immediately frozen in liquid nitrogen . the sample was then transferred to a low temperature field emission scanning electron microscope ( lt - fesem , jeol jsm 6300f ) equipped with an oxford cryochamber . after a light coating with argon gas the samples were observed and pictures were taken with a digital camera . [ 0143 ] fig1 . a diagram showing the microspore embryogenesis system we used to identify genes involved in embryogenesis . late uni - cellular microspores and early bi - cellular pollen isolated from b . napus ‘ topas ’ developed into embryos when cultured at 32 ° c ., while the same population of cells continued gamethophytic developement into mature pollen when cultured at 18 ° c . embryo or pollen materials can be harvested at different stages from these two conditions for rna isolation . [ 0144 ] fig2 . identification of the llp1 clone using differential display technique . a portion of a differential display gel showing the presence of llp1 cdna in 10 - day and 16 - day microspore - derived embryos . the rna samples were prepared from the following materials : microspores cultured for 8 hr at 18 ° c . ( 8 h 18 ° c . ); microspores cultured for 8 hr at 32 ° c . ( 8 h 32 ° c . ); microspores that were heat - shock treated at 42 ° c . for 45 minutes ( no embryos will be produced from such treatment , 45 ′ 42 ° c . ); note that , among these 9 rna samples , the llp1 signal ( indicated by an arrow ) was only seen in the lanes where the rnas were isolated from microspore - derived embryos after 10 and 16 days culture . [ 0155 ] fig3 the cdna and protein sequence of llp1 . the top strand shows the cdna isolated from a cdna library of brassica napus “ topas ” , and the bottom strand shows the fragment isolated originally by dd - pcr . the coding region together with the amino acid sequence was underlined , the signal peptide is double underlined , and the llp boxmotif boxed . [ 0156 ] fig4 northern blot hybridization showing the expression of llp1 gene in different organs and tissues of brassica napus “ topas ” . total rnas were isolated from tissues marked above the gel and hybridised with labelled llp1 fragment from dd - pcr . [ 0157 ] fig5 expression of llp1 gene during embryo and seed development in arabidopsis thaliana . a . diagrammatic drawing shows the expression pattern of the llp1 gene , as revealed by llp1 promoter :: gus fusion . b - f . gus staining of a late globular stage ( b ) and a heart - shape stage ( c ), cotyledonary stage ( d ) embryos and mature seed ( e ) and seed coat after germination ( f ). these results were obtained by gus staining of transgenic plants carrying llp1 promoter :: gus fusion construct . the llp1 gene is expressed firstly in a late globular embryo ( as marked in red ) and restricted to the top of the cotyledons ( as showed in c ) and later to the edge of the cotyledon at the torpedo stage . in cotyledonary embryos the expression is restricted to the base of the cotyledon , not in the apical meristem . the expression was switched off in the embryo thereafter . in mature and germinating seeds , the expression is restricted to the remaining endosperm ( also called aleurone layer , e and f ). [ 0161 ] fig6 llp1 promoter activity in the seedling stage ( 10 days after germination ). the llp1 gene is not expressed in young seedling within 5 days of germination . in 10 - day old seedlings , the llp1 gene starts to express in the axillary buds ( a ) and roots with well - established root hairs ( b ). such expression was excluded from the epidermal layer and the roothair . note that no expression was seen in the hypocotyl ( b ) and the newly formed side roots ( c ). [ 0166 ] fig7 the llp1 promoter activities in roots . the staining was carried out in seedlings 25 days after seed germination . a - e . a series pictures taken from one root at different positions . the expression of the llp1 gene is absence in the root - tip ( e ), highest in the root hair region ( d ) and gradually restricted to the vascular bundles ( c and d ) and disappeared in mature roots ( a ). f . a diagrammatic representation of the llp1 expression in the root system , as indicated in red . g . transverse section through the upper part of the root hair region indicating that the expression is mainly in the vascular system . [ 0170 ] fig8 llp1 promoter activities in the axillary buds and the inflorescence ( 25 days after seed germination ). a . longitudinal section through a young axillary bud revealing the expression of the llp1 gene is only in the periphery of the apical meristem . b . a developing axillary bud showing the promoter activity in the leaf primordia but not in the central meristem . c . llp1 gene is not expressed in mature leaves and stems . d . young flower buds showing the llp1 expression in the region between sepal and carpel primordia in young flower buds and then in the stigmatic cells . these cells form a two - lip structure at the beginning and a ring at the later stage . [ 0175 ] fig9 changes of branching patterns in arabidopsis thaliana “ c24 ” induced by the over - expression of the llp1 gene under the control of 35s promoter . a . electron microscopy photography showing a wild type stem with one shoot normally formed from each axillary bud . b . electron microscopy photography showing 3 inflorescences were formed from one axillary bud . c . at the later stage of plant development , more than 6 shoots could be seen from one axillary bud . [ 0179 ] fig1 male sterility and pin - shaped pistil induced by the over - expression of llp1 gene in arabidopsis thaliana “ c24 ”. b . flower from a 35s : llp1 transgenic plant showing the anther without viable pollen grains and pin - shaped pistil . some flower organs have been removed when the electron microscopy materials were prepared . [ 0184 ] fig1 defects in vascular development induced by over - expression of llp1 gene in arabidopsis thaliana “ c 24 ”. b , c . flower from llp1 over - expression plants showing the failure of xylem connection between flower and main stem . [ 0187 ] fig1 . llp genes in arabidopsis thaliana genome . peptide alignment of the llp genes identified from arabidopsis genome . in total 19 llp genes ( 1 - 19 ) have been found . all peptides encoded by these llp genes have an n - terminal signal peptide and a c - terminal conserved llp box . clv3 and three other llp proteins have a longer c - terminal span of sequences . [ 0188 ] fig1 llp genes identified in higher plants . alignment of llp proteins identified from arabidopsis and other higher plants . species with llp genes include arabidopsis , tomato , maize , soybean , medicago , and rice . the conserved llp box is . highlighted in color . maize esr proteins have longer c - terminal span after the llp box . [ 0190 ] fig1 . phylogenetic tree for all arabidopsis thaliana proteins that have a c - terminal llp boxmotif . [ 0192 ] fig1 . atllp1 : located on chromosome 3 , bac p1 clone muj8 accession b028621 ( 64541 until 65813 ) from arabidopsis thaliana [ 0193 ] fig1 . atllp11 : located on chromosome 3 , on bag clone p1 mfj20 accession ab026644 ( 76090 until 74701 ) from arabidopsis thaliana . [ 0194 ] fig1 . atllp12 : located on chromosome 5 , on bag clone p1 mxc9 accession b007727 ( 64512 until 66555 ) from arabidopsis thaliana . [ 0195 ] fig2 . atllp5 : located on chromosome 3 , on bag clone p1 mpe11 accession ab023041 ( 28993 until 27277 ) from arabidopsis thaliana . [ 0196 ] fig2 . atllp2 : located on chromosome 1 , on bac clone f14k14 accession ac011914 ( 54858 until 56409 ) from arabidopsis thaliana . [ 0197 ] fig2 . atllp7 : located on chromosome 5 , on bag clone p1 mxk3 accession ab019236 ( 2356 until 3738 ) from arabidopsis thaliana . [ 0198 ] fig2 over - expression of llp1 in arabidopsis leads to the consumption of root meristem b ) a llp1 over - expression seedling ( same age as in a ) shows the reduced growth in root . note the root hairs formed in the short root . c ) a close observation of root from a wt plant showing the normal root morphology . d ) roots from a llp1 over - expression plant showing the short and then root with the root hairs formed toward the tip ( same magnification as in d ). e ) wt root cleared with hoyer and observed with a dic microscope to showed the wt root morphology . f ) a root from a 7 - day old plant showing the reduced length of the root meristem and the elongation zone . g ) a root from a 10 - day old seeding showed the further reduction of root meristem and the elongation zone . the vascular bundle was indicated by an arrowhead . h ) a root from a 14 - day old seedling showed the disappearance of root meristem and the elongation zone . the vascular bundle ( indicated by an arrowhead ) was formed all the way to the central cell region . a ) the phenotype of the llp2 overexpression plant . note that no seeds have been produced from the 3 - month old arabidopsis . b ) close - up observation showing few flowers could be formed occasionally , but no seeds can be produced . c ) the flower formed in the llp2 over - expression plant produces 2 stamen and no pistil . no pollen was released from the anther . so such plant is both male and female sterile . d ) inflorescence meristem ( indicated by an arrow ) was terminated after producing 1 - 2 abnormal flowers ( removed to expose the meristem ). a ) three independent transformants ( to generation ) showing different degree ( light , medium and severe ) of phenotypes . plants with “ medium ” or “ severe ” phenotypes produce little or no seeds for further analysis , although the vegetative growth was normal . b ) and c ) progeny analysis of the “ light ” phenotype plants in the t1 generation . two types of phenotype were observed : sterility ( a ) and retarded growth ( b ). b ) genetic analysis showed that the sterile phenotype is a dominant trait in the t1 generation . a few wt plants obtained from segregation were removed . c ) a family of t1 plants shows the phenotype of retarded vegetative and reproductive growth . only few siliques were produced from each plant . the rosette leaves were also smaller . a few wt plants obtained from segregation were removed . [ 0215 ] fig2 the expression pattern of llp12 gene in arabidopsis . the result was obtained by analysis of llp12 promoter :: gus transgenic plants . a ) llp12 gene was expressed in the junction region of the roots . the expression was limited to the central vascular bundle . c ) diagrammatic drawing to indicate the llp1 expression in the inflorescence . the expression was seen only in the pedicel ( junction between main stem and the flower ) and the anther . [ 0219 ] fig2 the phenotype of llp12 over - expression of pants in the to generation . in both to transgenic plants showed here , the primary shoots were stopped early and multiple side shoots were formed afterward ( a and b ). the plants have very thin and short inflorescence , with no ( b ) or a few seeds ( a ) produced . the reduced seedset seemed to be caused by male sterility since seeds can be produced when cross - pollinated with wt pollen . flower development was normal , [ 0220 ] fig2 the phenotype of llp12 over - expression plants in the t1 generation . b ) llp12 over - expression plants 20 days after generation , showing the suppression of growth in both shoots and roots . c ) segregation of llp12 over - expression plants in the t1 generation , showing the few wt plants ( indicated by arrows ) were produced from the single insertion line . [ 0225 ] fig2 the over - expression of llp12 leads to male sterile phenotype and changes in flower positioning . a ) inflorescence of a llp12 over - expression plant showing the terminal position of flower and the side position of the inflorescence , which is a reverse of the wt morphology . b ) the changes in flower positioning can also been seen when the siliques were formed . note that the pedicel was also shorter . [ 0228 ] fig3 the over - expression of llp12 leads to reduced seedset and growth suppression . the apicla dominance also lost in these two transgenic lines ( t1 generation ). the wt plants were removed from the segregation population in the top figure , but not from the bottom figure ( indicated ). [ 0229 ] fig3 over - expression of llp12 anti - sense leads to plants with soft and short stems . a ) a plant at to generation showing short inflorescence with few siliques were produced ( a 3 - month old plant ). b ) plants in t1 generation showed few siliques were produced from each plant ( 1 month old ). c ) soft stem was a dominant trait in the segregation population . [ 0233 ] fig3 map position of 19 llp genes in fully sequenced arabidopsis genome . note the large cluster of genes observed at the bottom of chromosome 1 and nothing on chromosome 4 . none of these 19 genes have been annotated by the genome - sequencing project . taacatgctt cctttatttt agtgcacccg tag ctt gta tat aaa att tta tcc 54 aca caa att cct ttt gct cct ttt atc aaa cgt cgc tat ttg tat cag 102 tct acc tat ctg ctt tca tat tgt atc tcc cgc agc caa aca aaa act 150 ttt ttt ttg aca aag ata gaa cat gaa gat caa gag ttt gat att ggc 198 ttc ctc ttt tct gat tct tgc ctt cat tca tca ctc aga atc agc ttc 246 att tcg gag ttt gct gat gaa gaa tgg att gta cga aga aga aga agc 294 aaa aat tct att ggg cga ctc caa aga aac gat aac taattctaca 340 leu val tyr lys ile leu ser thr gln ile pro phe ala pro phe ile caaagatag aac atg aag atc aag agt ttg atg ttg gct tcc tct ttt ctg 51 att ctt gcc ttc att cat cac tca gaa tca gct tca ttt cgg agt ttg 99 ctg atg aag aat gta ttg tac gaa caa gaa gaa gca aaa att cta ttg 147 ggc gac tcc aaa gaa acg ata act aat tct aca gct ttg gag tct aaa 195 cgg ata att ccg acg ggt cca aat cca ctt cac aac agg taaatttgat 244 lys asn val leu tyr glu gln glu glu ala lys ile leu leu gly asp leu thr ile pro met thr arg his gln ser thr ser met val ala pro phe lys arg val leu leu glu ser ser val pro ala ser ser thr met arg glu phe gly asn asp ala his glu val pro ser gly pro asn pro pro pro pro pro thr glu ile asp gln arg tyr gly val glu lys arg cys asp ser phe ile arg pro tyr ala arg ser met cys ile glu leu met gly thr ile ala asn ser ser ala leu asp ser lys arg val ile met ala ser phe lys leu trp val cys leu ile leu leu leu leu glu phe ser val his gln cys arg pro leu val ala glu glu ser pro ser glu glu glu lys arg ser ile pro thr gly pro asn pro leu his asn met thr thr thr glu glu phe gln arg leu ser phe asp gly lys arg ile leu ser glu val thr ala asp lys lys tyr asp arg ile tyr gly met ala thr thr arg val ser his val leu gly phe leu leu trp ile tyr tyr arg pro gly arg arg 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