Patent Application: US-46657203-A

Abstract:
the invention relates to a method to modulate plant growth or development . the invention provides a method for modulating a developmental transition of a plant comprising modulating expression of a ring - h2 protein or functional fragment thereof in said plant or parts thereof .

Description:
tulip bulbs were harvested in the summer ( july ) and stored at 20 ° c . until at least two weeks after stage g ( september ). all storage treatments were performed in dark ventilated rooms . bulbs were then either transferred to 5 ° c . for low temperature treatment , or to 17 ° c . for the differential display analysis , after transfer to 5 ° c . or 17 ° c ., samples were taken after 4 , 8 and 12 weeks . the same was done in another year for the northern blot analysis of differential expression during temperature treatment . for the northern blot analysis of the different organs after planting , bulbs were transferred to 17 ° c . in september until the end of december and then transferred to 5 ° c . for 12 weeks . after planting and growing at 23 ° c . ( 12 hr light / 12 hr dark ), organ samples were taken at the indicated days after planting . all samples were frozen in liquid nitrogen and stored at − 80 ° c . or freeze dried . rna was isolated using ctab as described [ 3 ]. northern blots were performed using formaldehyde / agarose gels as described [ 1 ]. hybridization was performed at 68 ° c . using 50 % formamide buffer , and an α 32 p - ctp labeled rna probe . differential display pcr ( ddrt - pcr ) was performed as described [ 2 ]. the differential band was excised , and reamplified using the same conditions as during the ddrt - pcr , except that dntp concentration was 100 μm . the pcr product was cloned . all cloning of pcr products was in the pgem - t vector ( promega , madison usa ). after sequencing , two nested 5 ′ race ( rapid amplification of cdna ends ) reactions were performed using the marathon kit ( clontech , palo alto usa ) with fragment specific primers . the resulting fragment was cloned and sequenced . the total fragment was now reamplified and cloned using new primers from the 5 ′- end ( 5 ′- gtcgtcggcttcccctccgccaag - 3 ) and 3 ′- end ( 5 ′- tttaaccacaatagctcattgcaaggcttc - 3 )′ and proofreading enzyme ( klentaq , clontech ). two clones were sequenced on both strands and found to be identical . requested est clones were also sequenced on both strands . sequencing was performed on an automated sequencer ( alfexpress , amersham pharmacia biotech , uppsala , sweden ), using kits recommended by manufacturer . sequences were analyzed using dnasis ( hitachi software , olivet , france ) and generunner ( hastings software inc , hastings on hudson , usa ) software packages . database searches were performed using the ncbi blast server ( http :// www . ncbi . nlm . nih . gov / blast /). multiple alignments were performed at ebi clustalw server ( http :// www . ebi . ac . uk / clustalw /), using standard settings . alignments were shaded using boxshade 3 . 2 ( isrec , epalinges s / lausanne , switzerland ). using differential display a screening was performed for mrna &# 39 ; s expressed at a higher level in the bottom internode of dry stored tulip bulbs at 5 ° c . compared to those stored at 17 ° c . a 218 bp fragment was found to be differential and was cloned . because ddrt - pcr fragments often derive from the non - coding 3 ′- end , a 5 ′- race reaction was performed . the resulting fragment was sequenced and new 5 ′- and 3 ′- primers were designed to reamplify the full - length cdna using proofreading enzyme . the resulting 1222 bp clone contains an open reading frame of 327 amino acids with a predicted molecular mass of 36 . 7 kd . the cdna is either full length or close to full length because there is an in frame stop codon preceding the predicted start codon . the transcript size was also confirmed on a northern blot ( not shown ). the encoded acidic protein ( isoelectric point 5 . 02 ) has as most significant feature a c - terminal ring - h2 domain , a variant ring - finger domain . it is predicted to be targeted to the cytoplasm ( www psort - server : http :// psort . nibb . ac . jp : 8800 /). the protein will be further referred to as tgring1 . database searches revealed two partially sequenced est - sequences and one published arabidopsis sequence , cip8 [ cop - 1 interacting protein 8 ; [ 13 ]] with significant homology beyond the ring - h2 domain . the est - clones , rice est c10402 and arabidopsis est tai386 , were requested and full length sequenced . recently , the sequence of the arabidopsis est tai386 sequence has been published as aip2 , an abi3 interacting protein [ 8 ], and will be further referred to as such . the rice est will be further referred to as osring1 . a multiple alignment shows that the two est sequences are highly homologous to the tulip sequence over the whole length of the protein . osring1 shows 58 % identity and 72 % similarity over 300 amino acids , and aip2 51 % and 64 % respectively over 301 amino acids . the homology of cip8 to tgring1 is highest in the c - terminal part , but extends beyond the ring - h2 domain ( fig1 a ). further database research revealed that , although many sequences from every eukaryotic phylum contain the ring - h2 domain , some contain a ring - h2 domain with a conserved motif consisting of 25 - 40 amino acids directly n - terminal to the ring - h2 domain . these extended ring - h2 domains are found in sequences from plant , animal and viral origin ( fig1 b ). the expression level of tgring1 in bottom internodes is higher during the low temperature treatment compared to the control treatment ( fig2 a ). it is higher in the earlier part of the treatment than in later stages of the treatment . tgring1 expression was examined in different tissues after planting of the bulbs . expression in the internodes is examined at the moment when they just start to elongate rapidly and expression in the flower when it shows the first colouring . roots and leaves show no rapid growth phase , so expression is examined at an arbitrary moment ( fig2 b ). the highest expression is detected in flower tissue and the lowest in the leaves . we showed that one of the underlying mechanisms of low temperature induced stem elongation in tulips is a gradual change in the sensitivity for auxin in the bottom internodes over the course a low temperature treatment of a few months . this was shown by an increase in auxin induced internodal elongation and auxin induced gene expression of primary auxin response genes , after longer periods of low temperature treatment . by using differential display we have isolated a cdna clone that is expressed at higher levels during the low temperature treatment , as compared to a control treatment . it codes for a protein with a c - terminal ring - h2 domain . although this is a feature shared by a lot of proteins , both in arabidopsis and other species , only two arabidopsis sequences showed homology over the whole length of tgring1 . one of these , aip2 , has a sufficiently high homology to be a true homologue . this is also true for rice osring1 , which has slightly higher identity and similarity because it is of monocotyledonous origin , as tgring1 . the arabidopsis cip8 is a more distantly related protein . the extended ring - h2 domain shared by these four proteins is conserved beyond the plant kingdom , which indicates a special function for this domain . the arabidopsis proteins aip2 and cip8 are isolated as proteins interacting with developmental regulators . cip8 interacts with cop1 through specific interaction of the ring - h2 domain of cip8 and the ring - finger domain of cop1 [ 13 ]. the interaction of aip2 with abi3 is mediated by the c - terminal half of aip2 , containing the ring - h2 domain [ 8 ]. but surprisingly , it interacts with the b2 and b3 domains of abi3 , which do not contain a ring - finger domain . so either the c - terminal part of aip2 that does not contain the ring - h2 domain interacts with abi3 , or the ring - h2 domains can interact with different domains with no apparent sequence homology , despite the fact that the ring - h2 domains are highly similar in sequence ( 43 % identity and 71 % similarity between the ring - h2 domains of aip2 and cip8 ). low temperature induced stem elongation can be viewed as a form of dormancy breaking . stratification , vernalisation and dormancy breaking in tree buds are similar processes driven by low temperature . interesting now is that the tgring1 arabidopsis homologue aip2 is an abi3 interacting factor . the arabidopsis abscisic acid - insensitive3 ( abi3 ) mutant was originally isolated for its ability to germinate in the presence of inhibiting concentrations of abscisic acid ( aba ; [ 7 ]) and has a role in embryo maturation and dormancy maintenance [ 9 ]. recent publications have shown that abi3 also has a role in vegetative quiescence processes . the abi3 - 4 mutant shows a rapid induction of flowering and abi3 is expressed in the apex of dark grown seedlings [ 8 ; 11 ; 12 ]. another recent publication proposes a function for the ring finger domain in targeted ubiquitin - mediated proteolysis . ring finger proteins act therein as e3 ubiquitin protein ligases [ 6 ]. taken all these things together we show here that the low temperature induced tgring1 expression has a role in targeting the tulip homologue of abi3 for proteolysis , and thereby removing the state of dormancy , allowing the plant to grow under favourable conditions . analysis of aip2 in arabidopsis during processes as stratification and vernalization and its influence on abi3 protein levels are ways to further show this phenomenon , considering that the fact remains that two totally different approaches to analyze similar phenomena as seed dormancy and low temperature induced flowering in tulip result in the cloning of homologous proteins having the above described ring - h2 motif . determine the expression pattern of the arabidopsis homologue aip2 of the cloned tulip tgring1 during seed stratification by low temperature . dry arabidopsis thaliana seeds of the cape verdian ecotype ( cvi , planting date 21 june , harvested november 1 , start experiment november 11 , all in 2001 ) were sown on wet filter paper and either put in the dark at 20 ° c . or at 4 ° c . for 1 , 3 , 5 or 7 days . per treatment , a part of the seeds was after treatment transferred to 20 ° c . in the light ( 16 hrs light / 8 hrs dark ) to determine germination percentage ; the rest of the seeds were frozen in liquid nitrogen and stored at − 80 ° c . for rna isolation . germination percentage was determined 7 days after seeds were placed at 20 ° in the light . rna extraction and northernblotting was performed as described for the experiments on tulip . [ 0055 ] fig4 a and b show the expression level of aip2 after the indicated amount of days of treatment . the aip2 probe hybridized with a single band of approximately 1200 bp . the expression level is strongly increased 1 day after incubation at 4 ° c ., both compared to dry seeds ( twelvefold higher ) and seeds incubated at 20 ° c . ( fivefold higher ). the expression stays higher during the low temperature treatment , compared to the 20 ° c . treatment . [ 0056 ] fig3 c shows the germination percentage of the seeds after the different treatments . the seeds were not very dormant , yet the low temperature treatment increased the germination percentage , compared to seeds kept at 20 ° c . germination was 100 % after three or more days at 4 ° c . the results show that the arabidopsis aip2 mrna is strongly upregulated during cold stratification . this shows that the aip2 protein is not only conserved in sequence but also in function , moreover it is even functionally conserved in different developmental processes requiring low temperature induction , namely low temperature induced stem elongation in tulip and seed stratification in arabidopsis . the increased expression during cold stratification correlates with an increased germination percentage after the treatment . because aip2 interacts with abi3 and has a ring - h2 domain it is likely that the dormancy breaking occurs by proteolytic targeting of the abi3 protein via ubiquitination , thus removing the state of dormancy . aip2 would then act as a monomeric e3 ubiquitin ligase . by analogy the tulip tgring1 protein would have the same function in the process of low temperature induction of stem elongation , which can be seen as a form of dormancy breaking . this experiment shows that aip2 and homologous proteins are clear markers for dormancy breaking . it also shows the involvement of a ring domain protein in dormancy breaking in another process as low temperature induced stem elongation in tulips , namely cold seed stratification . a multiple alignment of tgring1 and homologous proteins from arabidopsis and rice . residues are shaded black when more than 50 % of the residues is identical and gray when more than 50 % of the residues is similar . fully conserved residues are indicated by an asterisk , conserved substitutions by a colon , and semi conserved substitutions by a dot . the ring - h2 domain is underlined , and the n - terminal extension of the ring - h2 domain is underlined by a dashed line . b multiple alignment of the extended ring - h2 domains of proteins from animal , plant and viral origin . shading is as indicated for a . species are indicated as follows : mm is mus musculus ( mouse ), rr is rattus rattus ( rat ), at is arabidopsis thaliana , os is oryza sativa ( rice ), hs is homo sapiens ( human ), tg is tulipa gesneriana and hh is human herpesvirus . database accession numbers are : praja1 np — 032879 , ndap1 baa06979 , rhc2a aac69860 , os329 baa88184 , hs311 baa91254 and icpo p28284 . a expression of tgring - 1 in the bottom internode of tulip during low temperature treatment ( 5 ° c .) compared to a control treatment ( 17 ° c .). b expression of tgring - 1 in different tissues after planting . as a reference , the expression level in the bottom internode at day 0 after planting is shown . this expression level is the same as that at week 12 of the 5 ° c . treatment in fig2 a ( different exposure times are used for fig2 a and fig2 b ). the other internode samples were taken at the moment that they started to grow rapidly : leaves and roots show no exponential growth phase and samples were taken at an arbitrary moment ; flower tissue was sampled when colouring started . 25 micrograms of total rna was used per lane . equal loading was checked by ethidium bromide staining ( not shown ). the predicted 327 amino acid open reading frame is coded for by bases 104 - 1182 and is indicated above the nucleic acid sequence . the bases coding for the ring - h2 domain are underlined . expression of aip2 during treatment of imbibed seeds at either 4 ° c . or 20 ° c . an rna probe was used complementary to bp 665 - 1120 of the aip2 mrna sequence ( genbank accesion ath251087 ). the band corresponds to a size of approximately 1200 bp . 8 μg of total rna was loaded per lane ; the bottom panel shows a methylene blue staining of the blot to confirm equal loading . ds indicates dry seeds . 1 . ausubel , f , brent , r , kingston , r e , moore , d d , seidman , j g , smith , j a , struhl , k : short protocols in molecular biology . john wiley & amp ; sons , new york ( 1995 ). 2 . bauer , d , muller , h , reich , j , riedel , h , ahrenkiel , v , warthoe , p , strauss , m : identification of differentially expressed mrna species by an improved display technique ( ddrt - pcr ). nucleic acids res . 21 : 4272 - 4280 ( 1993 ) 3 . chang , s , puryear , j , cairney , j : a simple and efficient method for isolating rna from pine trees . plant molecular biology reporter 11 : 114 - 117 ( 1993 ). 4 . de hertogh , a a , le nard , m : tulipa . in : de hertogh , a . a and le nard , m . 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