to identify lrl - related ( group xi basic helix loop helix ) gene sequences from early diverging land plants and streptophyte algae , we performed blast searches on marchantia polymorpha  and chara braunii sequences using arabidopsis thaliana  atlrl sequences as queries . single transcripts , cb_bhlhtranscript1 ( genbank accession number kx037431 ) and mp_bhlhtranscript1 ( kx037432 ) , were identified in each species encoding proteins with basic loop helix ( bhlh ) and lrl domains similar to those found in pplrl1 of physcomitrella patens and atlrl3 of a.  thaliana ( figure  s1 ) and were therefore designated mplrl and cblrl , respectively . these genes are most likely single - copy genes in c.  braunii and m.  polymorpha because a single band was detected in southern blots of gdna hybridized with a probe containing the lrl domain sequence ( figure  s1 ) . in flowering land plants , lrl transcription factors ( tfs ) form three well - supported clades ( xia , xib , and xic , termed here class i , ii , and iii ) . comparison of mplrl with other lrl sequences demonstrated that mplrl and pplrl are placed outside these three clades suggesting that lrl tfs diversified after the divergence of bryophytes and flowering plants ( figure  s1 ) . the cblrl is placed among the  class i lrl sequences on a long branch ( figure  s1 ) . no lrl - related sequences were identified in chlamydomonas reinhardtii or ostreococcus tauri or in the filamentous streptophyte klebsomidium flaccidum suggesting that lrl tfs evolved among complex streptophyte algae . taken together , these data indicate that the lrl proteins constitute a highly conserved transcription factor family , which diversified during streptophyte evolution from a most likely single - copy gene in the most ancestral groups of charales and liverworts to a gene family with several members belonging to different monophyletic clades in flowering plants . to assess whether diversification of the lrl gene family was accompanied by changes in gene expression , we compared expression patterns of lrl genes in c.  braunii , m.  polymorpha and a.  thaliana . in situ hybridization in c.  braunii showed that lrl mrna was present in most tissues of the plant ; cblrl mrna was detected in the nodes , internodes , and the oogonium surrounding the egg cell and zygotes ( figures 1a , 1 g , and s1 ) . in m. polymorpha , highest levels of mplrl mrna were detected in the meristematic notch by in  situ hybridization ( figures 1b , 1c , and s1 ) . highest fluorescence levels in the meristematic notch was observed in plants transformed with promplrl : nls-3xyfp ( figures 1d , 1h , and 1i ) indicating that the promoter was most strongly active in the same cells in which the mrna was detected . lower levels of yfp fluorescence were detected throughout the thallus ( figures 1e , 1j , 1f , 1k , and s1 ) . the relatively high expression in the notch and lower expression in the thallus is different from what is observed with ubiquitously expressed reporters ( compare figure  1d with figure  s1j ) , indicating that the mplrl expression heterogeneity is not caused by differences in cell densities along the thallus . together , these data indicate that mplrl is more highly expressed in the vicinity of the meristem than elsewhere in the thallus . this preferential expression in some domains is also observed in another early diverging land plant , the moss p.  patens . we determined the spatial expression pattern of each of the five a.  thaliana lrl genes using promoter : yfp fusions . the fluorescence patterns revealed that each of the promoters is active in distinct though overlapping domains . summed together these expression domains include cells of most tissue types ( figure  s2 ; summary in figure  1r ) . in the root , the differences between the atlrl gene expression patterns were most pronounced in the  root apical meristem ( ram ) and elongation zone ( ez ; figures  1l1p ) . the promoters of atlrl1 and atlrl4 were active early in the development of the provascular tissue and  endodermis , respectively ( figures 1l , 1l , o , and o ) . proatlrl3 was active in every cell and tissue ( figures 1n and 1n ) ; neither proatlrl2 nor proatlrl5 were active in the ram  ( figures 1m and 1p ) . later , in the root - hair zone ( rhz ) , proatlrl1 and proatlrl3 were active in every cell , while the proatlrl2 was active in most cells except pericycle and endodermis ( figures 1l1n and 1q ) . proatlrl4 was active in all tissues except the epidermis in the root hair zone , whereas proatlrl5 activity was detected in all tissues ( figures 1o1q ) . taken together , these data indicate that single - copy lrl genes are broadly expressed in c.  braunii and m.  polymorpha plants , while the different a.  thaliana lrl genes are expressed in distinct but overlapping domains . this suggests that lrl genes function throughout the plant in streptophyte algae and became progressively more restricted to specific tissues in land plants . in derived land plant taxa , multiple lrl genes are expressed in distinct domains , suggesting that different genes have distinct though overlapping functions . to investigate whether changes in gene expression patterns reflect changes in gene function , we compared m.  polymorpha and a.  thaliana lrl gene function . we generated two different inducible artificial microrna ( amir ) constructs to reduce mplrl  activity   using the ethanol inducible alcr / alca system   in  which the induced expression could be monitored  with an  inducible alca : nls-3xcfp reporter on the same t - dna . this  construct , alca : amir - mplrl - alca : nls-3xcfp - proosact : alcr ( the expression pattern of proosact is  shown  in  figure  s1 ) , was transformed and two lines for each  amir - mplrl sequence were established amiri - mplrl - ind-1 and amiri - mplrl - ind-2 ; amirii - mplrl - ind-1 and amirii - mplrl - ind-2 . each line expressed cfp and developed a defective phenotype only upon ethanol induction ( 20% ethanol vapor ; figure  s3 ) . induction of the expression of the amir - mplrl micrornas in these lines caused growth defects in gemmae  vegetative propagules of m.  polymorpha  which we classified into five categories ( c1c5 ) : ( c1 ) wild - type like plants ; ( c2 ) plants with undifferentiated patches of tissue outgrowth on the dorsal thallus ; ( c3 ) plants with larger outgrowths on the dorsal thallus indicating the initiation of secondary , ectopic thallus ( figure  2b ) ; ( c4 ) plants with a fully grown ectopic secondary thallus ( figure  2c ) ; and ( c5 ) the entire dorsal side of the plant was covered with rhizoid - less callus like tissue ( figure  2d ) . using these categories , we quantified the phenotypic variation in one amiri - mplrl - ind-1 and one amirii - mplrl - ind-1 line . both lines developed a significant number of callus - like phenotypes ( c5 phenotype ) , but the frequency of the phenotypes depended on the amir - mplrl sequences used ( figure  s3 ) . amiri - mplrl - ind-1 plants generally form secondary thalli ( c4 phenotype ) , while up to 46.9% of meristems in lines carrying the amirii - mplrl - ind-1 construct developed callus like tissue ( c5 phenotype ) . all defects could be complemented by double - transforming the amirii - mplrl - ind-1 line with an amirii - mplrl - resistant mplrl cdna , mplrlres , driven by the constitutive proosact promoter . the majority of meristems that developed on two independent mplrl knockdown lines transformed with the mplrlres gene developed wild - type features ( compare figure  2f with figures 2 g and 2h ) . to independently determine the function of mplrl , we fused the mplrl cdna to the ear repressor domain srdx [ 15 , 16 ] and expressed this fusion under the control of proosact promoter . the growth of the dorsal thallus of proosact : mplrl - srdx plants was severely impaired , resulting in the development of plants without dorsal characteristics and prominent ventral scales without rhizoids ( figures 2k , 2l , and s3 ) . severe morphological defects developed in 2-week - old  gemma expressing the mplrl - srdx fusion using an ethanol inducible mplrl - srdx gene construct ( compare mplrl - srdxind-1 and mplrl - srdxind-2 in figure  s3 ) . these plants were morphologically similar to the severe c4 and c5 phenotypes observed in amirii - mplrl - ind-1 lines . taken together , the absence of rhizoids in the most severe phenotypes of the  amir - mplrl and mplrl - srdx lines indicates that mplrl positively regulates rhizoid development . these results indicate that mplrl is a general growth regulator and required for dorsal development in gemmae . to define the function of lrl genes in angiosperms , we generated gain- and loss - of - function lines for each class of lrl genes in a.  thaliana . angiosperm class i lrl genes are positive regulators of root hair development [ 11 , 12 ] ; atlrl1 - 2 atlrl3 - 1 double mutants initiate root hairs that do not elongate ( figure  3b ) . furthermore , longer root hairs develop in plants that constitutively overexpress atlrl1 , atlrl2 , or atlrl3 ( figures 3d3f , 3i , and s4 ) ; plants transformed with 35s : atlrl1 or 35s : atlrl3 developed root hairs that were 899   70 ( mean   sd ) m and 823   27  m ( figure  3d , 3f , and 3i ) and were longer than wild - type root hairs ( 474   47  m ; figure  3a ) . atlrl5 - 1 
 35s : atlrl4amir - i or atlrl5 - 1 
 35s : atlrl4amir - ii plants  were indistinguishable from wild - type despite a significant decrease in steady - state levels of atlrl4 and atlrl5 mrna levels ( figures 3c and s4 ) . however , root hairs did not develop on wild - type plants  transformed with either 35s : atlrl4 or 35s : atlrl5 gene constructs demonstrating that atlrl4 and atlrl5 repress root hair elongation ( figures 3 g , 3h , and 3i ) . we conclude that class i and class ii lrl genes have antagonistic functions in the regulation of root hair development . to test whether the class i and class ii lrl genes reciprocally regulate each other s expression , we determined the steady - state levels of all atlrl mrnas in lines overexpressing each of the lrl genes . we could not detect changes of atlrl4 or atlrl5 steady - state mrna levels in plants that overexpress atlrl1 , atlrl2 , or atlrl3 ( figure  s4 ) . by contrast , steady - state levels of atlrl3 mrna were significantly reduced in 35s : atlrl4 and 35s : atlrl5 backgrounds ( figures 3j and s4 ) . these data suggest that class ii lrl genes repress the expression of the class i atlrl3 gene . we conclude that the two classes of atlrl act antagonistically during root hair development and class ii lrl genes have the potential to act as transcriptional repressors . if lrl gene function was conserved after c.  braunii and m.  polymorpha diverged from a common ancestor , we predicted that expression of cblrl would suppress the phenotypic defect caused by decreased mplrl activity in mplrl knockdown plants . we transformed the inducible mplrl knockdown ( amirii - mplrl - ind-1 ) plant with cblrl cdna under the control of a constitutive promoter . while defective gemmae developed on 92.2% of the amirii - mplrl - ind-1 lines in inducing conditions ( figure  4h ) , the majority of gemmae in amirii - mplrl - ind-1 ; proosact : cblrl plants were wild - type ( 67.9% ; figure  4j ) . in contrast , transformation of the amirii - mplrl - ind-1 line with the proosact : nls - yfp  control construct did not restore wild - type development ( 1.7% developed wild - type phenotypes ; figure  4i ) . to determine whether relatively derived lrl genes can substitute for loss of mplrl gene function , we expressed the atlrl1 ( class i ) and atlrl4 ( class ii ) cdnas using the proosact promoter in the background with decreased mplrl activity . only 27.3% and 29.7% of proosact : atlrl1 ; amirii - mplrl - ind-1 and proosact : atlrl4 ; amirii - mplrl - ind-1 lines developed wild - type gemma ( figures 4k and 4l ) . this indicates that the a.  thaliana lrl genes can not complement the defect in  mplrl loss - of - function lines as effectively as cblrl . these data support the hypothesis that angiosperm lrl genes have functionally diverged since m.  polymorpha and a.  thaliana last shared a common ancestor . we tested the ability of cblrl and mplrl to restore root hair  development in the atlrl1 - 2 atlrl3 - 1 mutant background . wild - type root hair growth was restored when double mutants  were transformed with proatlrl1:atlrl1 and proatlrl3:atlrl3 ( figures 4b4c , positive controls ) . only partial restoration of root hair growth was observed in proatlrl1:cblrl ; atlrl1 - 2 atlrl3 - 1 or proatlrl1:mplrl ; atlrl1 - 2 atlrl3 - 1 or proatlrl3:mplrl ; atlrl1 - 2 atlrl3 - 1 lines ( figures 4e4f ) . this is consistent with the hypothesis that a.  thaliana lrl proteins have functionally diverged from lrl proteins in early diverging plants , although some aspects of the ancestral function have been conserved . since expression of atlrl4 negatively regulate root hair development , and both atlrl1 and atlrl3 positively regulate root hair development , we predicted that expression of atlrl4 in the atlrl1 or atlrl3 domains of  the atlrl1 - 2 atlrl3 - 1 double mutant would not restore growth . as predicted , expression of proatlrl1:atlrl4 or proatlrl3:atlrl4 in atlrl1 - 2 atlrl3 - 1 double mutants did not restore root hair elongation ( figures 4d and 4d ) . taken together , these data support the hypothesis that lrl proteins have diversified during the course of land plant evolution . a single lrl gene controls thallus and rhizoid development throughout the early diverging land plant m.  polymorpha , a function that partially has been conserved since c.  braunii and land plants diverged from a common ancestor . gene duplication followed by functional diversification gave rise to two classes of antagonistically acting proteins expressed in distinct but overlapping domains in relatively derived plants such as a.  thaliana . antagonistic regulation is critical in gene regulatory networks facilitating negative feedback or incoherent feedforward regulation . it is therefore conceivable that the  diversification of lrl gene function was an innovation that contributed robustness to the network , critical for the integration of internal signals with changing environmental conditions in derived groups of land plants .
summarystreptophytes colonized the land some time before 470 million years ago [ 1 , 2 , 3 ] . 
 the colonization coincided with an increase in morphological and cellular diversity [ 4 , 5 , 6 , 7 ] . 
 this increase in diversity is correlated with a proliferation in transcription factors encoded in genomes [ 8 , 9 , 10 ] . 
 this suggests that gene duplication and subsequent diversification of function was instrumental in the generation of land plant diversity . here , we investigate the diversification of the streptophyte - specific lotus japonicus roothairless like ( lrl ) transcription factor ( tf ) [ 11 , 12 ] subfamily of basic loop helix ( bhlh ) proteins by comparing gene function in early divergent and derived land plant species . 
 we report that the single marchantia polymorpha lrl gene acts as a general growth regulator required for rhizoid development , a function that has been partially conserved throughout multicellular streptophytes . 
 in contrast , the five relatively derived arabidopsis thaliana lrl genes comprise two antagonistically acting groups of differentially expressed genes . 
 the diversification of lrl genes accompanied the evolution of an antagonistic regulatory element controlling root hair development .