Patent Application: US-201514733653-A

Abstract:
described are methods of isolating genes involved in the process of asymmetric cell division . further disclosed are genes isolated with this method , and their use in controlling root formation , preferably lateral root formation .

Description:
in the lateral root inducible system , seeds ( col - 0 and slr - 1 ) were germinated on standard murashige and skoog media containing 10 μm n - naphtylphtalamic acid ( npa ) on vertically oriented square plates ( greiner labortechnik , frickenhausen , germany ) in a growth chamber under continuous light ( 110 μe · m - 2 · s - 1 par supplied by cool - white fluorescent tungsten tubes [ osram , münchen , germany ]) at 22 ° c . ( himanen et al ., 2002 ). seventy - two hours after germination ( 0 hour time point ), only those seedlings ( wild - type and mutant ) that made full contact with the medium were transferred to medium containing 10 μm naphtylacetic acid ( naa ) and harvested after two hours and six hours . these three time points were applied for both wild - type and mutant . a mock treatment was included for wild - type only , by transferring the seedlings to standard murashige and skoog medium without naa addition . for all time points , only the lateral root inducible segments were used for the analysis . for this purpose , the root apical meristem and hypocotyl were manually removed to minimize contamination with other cell types . all treatments were repeated . rna was extracted using the rn easy ® minikit ( qiagen ). rna quality and quantity were analyzed using rna 6000 nano lab chip kit ( agilent technologies , germany ). for microarray , 5 . 8 μg total rna was used . double - stranded cdna was synthesized with life technologies cdna synthesis kit . the double - stranded cdna was converted to biotin - labelled crna ( ambion mega script t7 in vitro transcription kit and biotin - containing ribonucleotides from enzo ( loxo gmbh )). fifteen μg of fragmented crna was used for hybridization to ath1 affymetrix ® gene - chips . the biotin - labelled rna was visualized with phycoerythrin - streptavidin labels . ath1 gene - chips ( affymetrix ) represent 22747 arabidopsis genes (˜ 85 % predicted genes in the arabidopsis genome ). the overall signal of the different chips was normalized using microarray suite 5 . 0 software ( affymetrix ). the raw data were exponentially distributed and were , therefore , 2 log - transformed before further statistics . statistical significance was analyzed via analysis of variance ( anova ) for every gene . this resulted in a p - value for three sources of variance : the effect of the time course , the effect of the genotype and the effect of their interaction . for the genome - wide transcript profiling , the stringency was increased to p & lt ; 0 . 001 . this is the equivalent of 23 false positive tests if 22747 tests are performed . at this level of significance , 3110 genes were flagged . there was a need to detect differences between the expression profiles in both genotypes , so a tool to optimally visualize these differences was required . this tool was obtained by merging time course data for both genotypes per time point . this merged dataset was subsequently treated as if it was a single time course ( repeated time points are indicated with *) ( 0 / 0 */ 2 / 2 */ 6 / 6 *). before clustering , an estimate of the predictive power of a clustering algorithm ( figure of merit ) was computed over a range of clusters . the lower the figure of merit , the higher the predictive power of the clustering will be ( yeung et al ., 2001 ). the number of clusters , for which the smallest increment did not result in a decrease of the figure of merit , was chosen as the optimal cluster number . all clustering computation was performed using tigr multi - experiment viewer 2 . 2 ( tigr . org webpage , 10 / 11 / 2003 ). each gene was related to two clusters , representing its average expression profile in both wild - type and mutant . the combinational potential was represented in a cross - table format with indication of the frequency of occurrence of each combination . as an indicator of differences between clusters , a color code was applied . for all clusters , the relative induction / reduction rates of the expression profiles between zero and 2 hours and between zero and 6 hours of the average profiles were compared to one another . if these relative induction / reduction rates differed two - fold or more at one of these levels of comparison , an orange or blue color was assigned to this cluster combination . if these relative induction / reduction rates differed at both levels two - fold or more , a red color was assigned to this cluster combination . a cluster was considered as up - regulated when the rate of induction of the expression level was stronger than two - fold for both intervals ( 0 - 2 and 0 - 6 ). only clusters 1 , 2 , 3 and 4 met these criteria . in the lateral root inducible system , seeds ( j0121 , plantsci . cam . ac . uk / haseloff / genecontrol / catalogues / jlines / record / record — 0 . html webpage ) were germinated ( himanen et al ., 2002 ). as described above , seedlings were harvested after 2 hours and 6 hours . for all time points , the roots were cut into small 0 . 5 mm fragments , and those segments were protoplasted according to birnbaum et al . ( 2003 , 2005 ). gfp - expressing cells were isolated on a fluorescence - activated cell sorter ( becton dickinson facsvantage ). the cells were sorted directly into lysis buffer ( qiagen rlt buffer ), mixed and immediately frozen at − 80 ° c . for later rna extraction . all treatments were repeated . standard affymetrix ® protocols for small samples were then used for amplifying , labeling and hybridizing rna samples ( wi . mit . edu / cmt / protocols / affysmlsamplproto . pdf webpage ). the hybridized crna was fragmented as described in the genechip ® expression analysis technical manual . the hybridization , washing and staining steps were performed according to the affymetric protocols ( wi . mit . edu / cmt / protocols / affymetrix % 20user % 20manual . pdf webpage ). the data were processed using a mixed model . this mixed - model analysis of variance was performed to identify genes differentially expressed between the various treatments ( chu et al ., 2002 , 2004 ). in this approach , a global normalization step was applied to minimize general array - level effects by centering the mean of the log 2 - transformed values to zero for each array ( chu et al ., 2002 ). outlier probes with values greater than two standard deviations from the probe - set mean were then removed . next , a mixed - model anova was applied to the transformed and centered intensity values obtained from the global normalization step . this gene model , which is based on that developed by chu et al . ( 2002 ), can be formalized as : log 2 ( pm jkl )= t j + p k + a l ( j ) + ε jkl where the pm variable refers to the output of the global normalization procedure for each gene , as described above . the symbols t , p , and a represent treatment , probe , and array effects , respectively . the array effect a l ( j ) is assumed to be a normally distributed random effect ( chu et al ., 2002 ). a standard error term ε jkl was also applied to this model . in addition , the indices j , k , and l represent the jth treatment , on the kth probe , and on the lth replicate ( chu et al ., 2002 ). the output of this model is the mean expression value for every gene , based on the global model , as well as a p - value from the gene - model for the probability of falsely rejecting the null hypothesis of no - differential expression ( α = 0 . 05 ). the global and gene models were run on a linux ® server with the statistical software sas ( version 8 . 2 ). grouping of the 1920 significantly differentially expressed genes coming out of the statistical analysis into 10 clusters , was done using tigr mev 3 . 0 . 3 ( saeed et al ., 2003 ). recently , an auxin - based lateral root inducible system was developed ( himanen et al ., 2002 ). based on this unique , in planta inducible system , a genome - wide transcript profiling was performed to identify key regulators of lateral root initiation . to facilitate the identification of those genes with a role in auxin signaling in relation to lateral root initiation , a mutant was included as a negative control . the mutant ( solitary root ) was mainly selected for its inability to form lateral roots and because the affected gene is involved in a known part of auxin signaling ( fukaki et al ., 2002 ). the comparison of wild - type and mutant in the lateral root inducible system is of fundamental importance to select genes involved in lateral root initiation , downstream of the protein affected in the mutant ( iaa14 / slr ). time points were chosen in such a way that allowed monitoring gene expression at different stages just prior to the first division in the pericycle . himanen et al . ( 2002 ) showed that this event occurs 8 to 10 hours after transfer to auxin - containing medium . the zero time point ( 72 hours npa ) is consistent with a g1 / s - blocked state , while six hours after transfer to auxin , pericycle cells adjacent to the xylem poles are nearly starting g2 / m transition . furthermore , the earliest auxin response in the root was visualized with a dr5 :: gus reported 1 . 5 to 2 hours after auxin treatment ( fig2 ). therefore , a time point ( 2 hours naa ) was included to represent this earliest auxin - modulated transcription . both wild - type and mutant ( slr - 1 ) were subjected to these treatments . additionally , a mock - treatment was included for wild - type to assess differential gene expression due to the transfer . all treatments were biologically repeated , adding to the statistical significance of the data . after normalization and transformation , the data were subjected to anova analysis . comparison of the previous limited transcript profiling ( on 4600 genes ) ( himanen et al ., unpublished results ) with the present one , clearly shows that our lateral root inducible system is highly reproducible , since 64 % of the differentially expressed genes were confirmed when checked at the same level of significance ( p & lt ; 0 . 005 ). in order to reduce the amount of false positives even further , a five - fold higher stringency ( p & lt ; 0 . 001 ) was applied than in the previous transcript profiling . at this high stringency level , 3110 genes were still differentially regulated . clustering of all data - points for both wild - type and solitary root separately did not meet the needs to assess the differences in expression profiles in both genotypes . in order to meet this criterion , the data for both genotypes were combined into one dataset . each gene was represented twice in the combined dataset , resulting in 6220 expression profiles . in order to estimate the optimal number of clusters , the figure of merit ( fom ) was computed for a range of clusters . the smallest fom was estimated at 14 clusters , representing the optimal number of clusters corresponding to the highest predictive power ( fig3 ). subsequently , all 6220 expression profiles were clustered into 14 clusters . in this way , two coordinates were assigned per gene , representing the expression profiles in both genotypes . all 196 ( 14 × 14 ) potential combinations are represented in fig4 , together with the absolute frequency of genes in each combination . differences between clusters are indicated through a color code . the genes indicated in red ( 305 ) represent the genes for which wild - type gene expression is always higher than in solitary root . genes of this kind , induced in wild - type and less in solitary root , are most likely involved in lateral root initiation . therefore , focus was on the genes , represented as clusters 1 , 2 , 3 and 4 in wild - type . in this way , the total number of significantly regulated genes ( 3110 ) was narrowed down to 266 (˜ 9 %) that might have a crucial role in lateral root initiation . a comparison of the percentages of genes belonging to a functional category before and after clustering according to matdb ( mips arabidopsis thaliana database ) nicely illustrates the effectiveness of the cross - table - based clustering ( table 1 ). the filtering procedure clearly resulted in an enrichment for genes related to cell cycle , rna processing , dna synthesis and signaling and development . these features confirm that the monitoring of cell cycle progression in the pericycle is possible using the lateral root inducible system . furthermore , a higher percentage of genes involved in transcriptional regulation were found , indicating that there is a general need for increased transcriptional activity . the percentage of unclassified and unknown genes remains at about the same level . moreover , a strong relative reduction of genes involved in stress , transport and metabolism was achieved through the applied selection criteria . in addition , the drop in the number of genes involved in transport might also be categorized as a drop in number of genes related to detoxification and stress responses . detailed examination of the selection reveals g1 / s and s - phase markers such as arath ; cycd3 ; 2 and arath ; cyca2 ; 4 . furthermore , the link to s - phase entry / progression is never far - off as there is a high representation of genes involved in dna replication and protein synthesis . this underlines the suitability of this approach to study auxin - mediated cell cycle regulation . as interesting as core cell cycle events can be , they require upstream signaling cascades such as auxin signaling . in this stringent selection , several genes were detected belonging to gene families with known roles in auxin signaling such as aux / iaas , arfs , atgh3s and an atsaur ( hagen and guilfoyle , 2000 ). different mutants in genes belonging to the aux / iaa gene family have lateral root phenotypes ( fukaki et al ., 2002 ; park et al ., 2002 ). their gene products act to repress the activity of arf transcription factor dimers ( leyser , 2002 ). recently , researchers gained insight into the function of atgh3 - gene products , through the analysis of activation tagged lines ( takase et al ., 2004 ). several of these gh3 proteins have been shown to adenylate plant hormones and based on their substrate specificity and protein structure , they are subdivided into three major classes ( staswick et al ., 2002 ). the members of group ii , such as atgh3 - 1 , atgh3 - 5 and atgh3 - 6 / dfl1 , can adenylate iaa , negatively regulating auxin activity ( takase et al ., 2004 ). as for the small auxin up rnas ( atsaur ), very little is known about their function in auxin response . however , their auxin inducibility has been reported for several years ( mcclure and guilfoyle , 1989 ). as transcription factors play central roles in patterning and development ( sabatini et al ., 2003 ), it is obvious that such genes in this selection ( 19 ) will be of particular importance in the signaling cascades during lateral root initiation ( table 1 ). most of the genes of this selection were recently shown to be specifically expressed in stele tissue ( including the pericycle ) by a transcript profiling study of the arabidopsis root tip ( birnbaum et al ., 2003 ), justifying the selection criteria used in the study . interestingly , two of the ap2 domain transcription factors belong to the same subclade . this implicates that it is likely that these genes have redundant functions . furthermore , there is one ap2 domain transcription factor that belongs to this same subclade of three genes , which is not represented on the microarray ( alonso et al ., 2003 ). it is hypothesized that this gene ( at4g27950 ) could also be functionally redundant to the two other members of this subclade . consequently , this gene was also added to the selection , bringing the final number of the selection to 20 genes . within the dataset , there are 15 transcription factors for which no role in auxin signaling has been suggested . as for a start of validation , it will be of primary interest to do a functional analysis on these transcription factors with respect to lateral root initiation . many of these transcription factors have great potential for involvement in lateral root development , as they have homologues for which a role in organ development has been reported . the abi3 gene was previously described as a seed - specific gene , but recently , it has been shown to have a role in auxin signaling and lateral root development ( brady et al ., 2003 ). also , ap2 and several homeobox genes have been shown to be involved in floral organ development , which implies that homologs have great potential to be essential in the development of other organs such as lateral roots ( carpenter and coen , 1990 ; maes et al ., 1999 ). interestingly , there is one transcription factor , myb 124 , for which the mutant has an aberrant stomatal development . as the result of the mutation , stomata with four guard cells are formed instead of two ( yang and sack , 1995 ). its up - regulation upon auxin treatment of the root implicates that the myb 124 gene product might have as crucial a role in the formative divisions in the pericycle ( lateral root initiation ) as it has in stomatal development . using lri as a model to genetically dissect the asymmetric cell division , it was investigated which cluster within the ten clusters contained the putative regulators of this type of division . first , it was analyzed which cluster is strongly linked with the g2 - to - m transition by verifying the expression profile during cell cycle progression using the genome - wide expression data for synchronized arabidopsis cell suspensions ( menges et al ., 2003 ). it was found that 48 % of the genes in cluster 3 peaked at the g2 - to - m transition . this is opposed to less than 10 % of the genes that peaked at this transition in all the other clusters . this is a strong overrepresentation of g2 - to - m - related genes within this cluster as compared to the other clusters . furthermore , this is 59 % of the g2 - to - m - specific genes present in the whole dataset . secondly , which cluster is potentially correlated with asymmetric cell division was analyzed . for this , protein sequences of genes were used that are assigned to the functional categories ( godatabase . org / cgi - bin / amigo / go . cgi webpage ) “ asymmetric cell division ” and / or “ establishment and / or maintenance of cell polarity ” in a wide variety of organisms i . e ., caenorhabditis elegans , drosophila melanogaster , schizosaccharomyces pombe , mouse . . . ). a protein blast analysis was performed with the protein sequences of various organisms and those arabidopsis protein sequences of the genes assigned to the different clusters . this resulted in an overrepresentation of 54 genes putatively correlated with “ asymmetric cell division ,” “ cell fate commitment ” and / or “ establishment and / or maintenance of cell polarity ” in cluster 3 . to further analyze the process of asymmetric cell division , focus was on , therefore , the 340 genes within cluster 3 . within this cluster , 25 % of the genes have been described ; the remaining 75 % is unknown , expressed , hypothetical or putative . in order to even further reduce the number of interesting candidates , those genes were subtracted of which involvement in normal cell division ( synchronized , dividing arabidopsis cell suspension cells ) is shown ( menges et al ., 2003 ). after that analysis , 190 candidates potentially involved in asymmetric cell division remained . it has been previously demonstrated that cell cycle progression in the pericycle is not sufficient for solitary root / iaa14 - mediated lateral root initiation in arabidopsis thaliana ( vanneste et al ., 2005 ). to support this finding , the root phenotype of transgenic lines over - expressing ( 35s ) cell cycle genes were analyzed . based on the cell cycle stage - specific expression profiles upon lateral root induction shown in himanen et al . ( 2002 ) and the dataset , cycd3 ; 1 ( g1 - to - s and g2 - to - m , dewitte and murray , 2003 ), e2fa / dpa ( g1 - to - s , de veylder et al ., 2002 ) and cdkb1 ; 1 ( g2 - to - m , boudolf et al ., 2004 ) were selected . the over - expression phenotype in 10 - day - old seedlings of all lines was analyzed and compared to wt ( fig5 ). none of the transgenic lines showed a significant increase in the lateral root density . the double transgenic over - expression line of e2fa / dpa even showed a strong decrease in the lateral root number compared to the wild - type col . also , over - expression of cdkb1 ; 1 resulted in fewer lateral roots . furthermore , in the case of cdkb1 ; 1 , the over - expression of dominant negative allele of cdkb1 ; 1 ( cdkb1 ; 1 . n161 ) ( boudolf et al ., 2004 ), resulted in a stronger decrease of the number of lateral roots . next , analysis was performed as to whether auxin ( naa ) application in combination with increased cell cycle gene expression could result in a higher number of lateral roots compared to the auxin or cell cycle alone . therefore , five - day - old seedlings of the above - mentioned transgenic arabidopsis lines of e2fa oe , dpa oeoe , cycd3 ; 1 oe and cdkb1 ; 1 oe were transferred to increasing concentrations of auxin ( 10 − 8 , 10 − 7 and 10 − 6 m of naa ) and , after another 5 days of growth , their ability to initiate lateral roots was analyzed . a significant increase was found in the cycd3 ; 1 oe opposed to the wild - type . similarly , the number of lrs / cm could be significantly increased in the e2fa / dpa oe transgenic line , until exceeding the wild - type number at high auxin concentration . even cdkb1 ; 1 oe exceeded the wt number upon auxin application , while this was not the case for the cdkb1 ; 1dn oe line . the above results indicate that stimulating the basic cell cycle machinery is not sufficient for de novo lateral root initiation , but when extra auxin is provided , the enhanced cell cycle competence can be exploited to produce new organs . this corroborates the suggestion by vanneste et al . ( 2005 ) that , next to cell cycle activation , another factor is required to specifically drive lateral root initiation . notwithstanding a putative function for cdkb1 ; 1 in lateral root initiation , cell cycle genes are clearly not the key regulators for lateral root initiation . hence , a search was performed for potential specific regulators of lateral root initiation via meta - analysis within the dataset . this meta - analysis was performed to reduce the number of genes from 1920 significant genes to 15 highly interesting candidates ( table 3 ). the analysis involved subsequent steps of overlapping and in - depth analysis of subsets of genes as described below . 1 ) affymetrix arabidopsis ath - 1 genome array ( 22758 genes ); 2 ) unique significantly differentially expressed genes ( 1920 ); 3 ) up - regulated genes in asymmetric cell division during lateral root initiation after selecting 1 cluster based on the following criteria ( 340 ) using in - depth analysis with functional categories terms : highest % g2 - m genes highest % genes involved in asymmetry highest % genes involved in polarity highest % genes involved in cell fate ; 4 ) genes potentially involved in cell fate and cell polarity ( 190 ) after subtracting the mitotic apparatus based on menges et al . ( 2003 ); 5 ) genes involved in auxin - induced cell fate and / or cell polarity in the xylem pole pericycle during lateral root initiation ( 15 ) after overlapping the remaining 190 genes with those lateral root initiation genes ( 913 ) depending on rapid slr / iaa14 degradation for normal auxin responsiveness , as derived from the cross - table ( fig4 ). as was determined earlier ( vanneste et al ., 2005 ), an important regulator mechanism for lateral root initiation is auxin signaling and transport . table 4 lists the genes involved in those events and demonstrates that most of them are early up -/ down - regulated . a number of genes have been shown to be involved in lateral root formation ( alf1 / rty / sur1 , celenza et al ., 1995 , king et al ., 1995 , boerjan et al ., 1995 ; dfl1 , nakazawa et al ., 2001 ) and for several aux / iaas and arfs , a role in lateral root initiation and / or formation was shown earlier ( iaa19 / msg2 , tatematsu et al ., 2004 ; arf19 , wilmoth et al ., 2004 ; iaa1 / axr5 , yang et al ., 2004 ; iaa3 / shy2 , tian and reed , 1999 ). for bdl / iaa12 , part of a pair of transcriptional regulators with mp / arf5 , the involvement in lateral root initiation was demonstrated . xylem pole pericycle - specific expression of a stabilized mutant version of the bdl protein in j0121xuas : bdl ( 0 . 0 ± 0 . 0 ) resulted in a lateral rootless phenotype , while the control lines col - 0 ( 3 . 0 ± 0 . 1 ), j0121 ( 4 . 1 ± 0 . 1 ), uas : bdl ( 3 . 1 ± 0 . 1 ), uas : bdl ( 2 . 8 ± 0 . 1 ) and j0121xuas : bdl ( 3 . 6 ± 0 . 1 ) displayed no reduction in the number of lateral roots / cm ( fig6 ). cyca2 ; 4 was identified as a putative important regulator of cell division during lateral root initiation ( vanneste et al ., 2005 ). however , overexpression of cyca2 ; 4 did not induce an increase in lateral roots ( similar to overexpression of other cell cycle genes ), while it did stimulate cell cycle progression as exemplified by a strong reduction of endoreduplication level in cotyledons . also , in knock - outs , no obvious changes in lateral root density were observed . but , cyca2 ; 4 belongs to a small gene family consisting of four members . combining mutations in various members of this family did result in dramatic reductions of lateral root density ( fig7 ). taken together , these data suggest that a2 - type cyclins are required , but not sufficient for lateral root initiation to occur . interestingly , cyca2 ; 4 — a core cell cycle gene — is retained in the list of genes after meta - analysis . unfortunately , the lack of lateral root phenotype in the overexpression lines might suggest that a combination of genes / factors is required to specifically drive asymmetric cell division and lateral root initiation . the most likely candidates that , when combined , will induce lateral root initiation are within the subset of 15 genes identified under example 8 . a number of salk t - dna insertion mutants from genes in various clusters were made homozygous and analyzed for their lateral root phenotype ( fig8 ). in the graph , bars of mutants with a significant increase or decrease in the lateral root number are colored green or red , respectively . the green or red box around part of the graph indicates genes from up - or down - regulated clusters , respectively . in addition to the lateral root phenotype , defects in other processes requiring asymmetric cell divisions were also detected , i . e ., stomata formation and embryogenesis . a number of promoter - gus / gfp fusions from genes in various clusters were made homozygous and analyzed in detail for their expression pattern ( fig9 ). mainly , the gus / gfp expression pattern was in agreement with the up - or down - regulation of the gene in the microarray dataset . for four genes , the expression pattern was analyzed in detail , and revealed a specific up - or down - regulation of the gus / gfp in the lateral root initiation site at the time of asymmetric cell division in agreement with the transcript level detected in the microarray ( fig1 ). brady s . m ., s . f . sarkar , d . bonetta , and p . mccourt ( 2003 ). the abscisic acid insensitive 3 ( abi3 ) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in arabidopsis . the plant journal 34 : 67 - 75 . carpenter r . and e . s . coen ( 1990 ). floral homeotic mutations produced by transposon - mutagenesis in antirrhinum majus . genes and development 4 : 1483 - 1493 . casero p . j ., i . casimiro , l . rodriguez - 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