BACKGROUND
transparent testa glabra  proteins were identified as regulators of trichome and seed development in plants .
ttgs are characterized by the presence of the wd <dig> motif , which refers to the conserved tryptophan  and aspartate  dipeptide and the length of approximately  <dig> amino acid residues in a protein sequence  <cit> .
this motif is present as a series of repeats to form protein interaction domain in a variety of proteins , including all ttgs so far identified except for arabidopsis thaliana atttg <dig>  which is a wrky transcription factor  <cit> .
as the wd <dig> domain has multiple modes for recruiting different substrates, wd40-containing proteins can serve as interchangeable substrate receptors and interact with diverse types of proteins  <cit> .
due to this characteristic, ttgs have the potential of regulating both development and defenses in plants  <cit> .
depending on natures of interacted proteins, ttgs differentiate their functions between development and defense regulations  <cit> .
in arabidopsis, atttg <dig> interacts with the bhlh transcription factor gl <dig> while recruiting myb gl <dig>  forming the wd40-bhlh-myb complex that acts to regulate trichome development  <cit> .
previously we demonstrated that wd <dig> enabled tobacco  ttg <dig>  to interact with para <dig>  <cit> , an elicitin protein produced by an oomycete pathogen and inducing hypersensitive cell death in the plant  <cit> .
the ntttg1-para <dig> interaction was essential for the induction of cell death first in leaf trichomes and then in mesophyll cells  <cit> .
ntttg <dig> is an analog of ntttg <dig> and both proteins share four wd <dig> repeats  <cit> .
recently we showed that ntttg <dig> suppressed tobacco resistance to pathogens by indirectly modulating subcellular localization of the npr <dig> protein  <cit> , which is a transcriptional regulator of defense responses to pathogens .
ntttg <dig> does not interact with npr <dig> but is able to sequester npr <dig> from the nucleus, thus preventing npr <dig> from transcriptional regulation of defense responses  <cit> .
these studies suggest that ttgs regulate specific processes of development or defenses by either directly or indirectly interacting with other proteins involved in the processes.
however, whether ntttg <dig> plays a role in development regulation was unclear.
plant development is subject to a complex regulation of phytohormones with auxin playing a significant role.
auxin mediates a stunning array of the development  <cit>  through the functions of auxin response factor  proteins in transcriptional regulation of auxin responses .
arf transcription factors constitute a phylogenic protein family  made of 10– <dig> members as identified so far in different plant species .
it is accepted that different members of the arf protein family target distinct types of auxin-responsive genes, such as those classified in the small auxin up rna , auxin/indole-acetic acid inducible , and gretchen hagen  <dig>  gene families .
however, only a few of auxin-responsive genes have been shown definitely as arf targets and to be regulated directly by specific arfs  <cit> .
in particular, little was known about the regulation of auxin responses in tobacco.
in the plant, only four auxin-responsive genes were identified, whereas, at least  <dig>   <dig>  and  <dig> members of aux/iaa <cit> , saur <cit> , and gh <dig> <cit>  gene families were characterized in other plants.
in tobacco, moreover, only arf <dig> <cit>  and arf <dig>  were cloned with full length cdna sequences while additional eleven arf homologs were identified as expressed sequence tags   <cit>  in contrast to the 10– <dig> members of the arf protein family already identified in other plants .
therefore, it is rational to conjecture that tobacco may possess more arfs and auxin-responsive genes than the currently known numbers.
in addition, there is as yet no study to characterize functions of arfs in tobacco.
this study was attempted to elucidate the role of ntttg <dig> in the growth and development of tobacco and to identify arf gene candidates associated with ntttg2-regulated growth and development.
we show that ntttg <dig> is required for the vegetative growth and the development of flowers and seeds.
we performed de novo assembly of the tobacco n. tabacum transcriptome and analyzed functional components in the transcriptome.
by using transcriptome information, we identified the unigenes assigned to auxin responses and representing putative arf genes in particular.
we present evidence that ntttg <dig> affects the expression of putative arf genes in the plant.
RESULTS
the repressive effect of ntttg <dig> silencing on the vegetative growth and the development of flowers and seeds in tobacco
to elucidate the roles of ntttg <dig> in the growth and development of tobacco n. tabacum, we generated the ntttg2-silencing line ttg2rnai <dig> under background of the n. tabacum variety nc <dig> and observed its growth and development in contrast to the wild-type  phenotype.
the transgenic nc <dig> line wt rfp was used as a control since it highly resembled wt in the development process  and biological productivity .
rfp is a red-fluorescent protein-encoding gene used in detecting subcellular localization of the protein  <cit>  but is not related to this study.
molecular and genetic characterizations of transgenic plants have been documented in a recently published article  <cit> .
in this study, we investigated the vegetative growth of wt rfp and ttg2rnai <dig> plants on murashige and skoog  agar medium and in pots.
we also analyzed the expression of the ntttg <dig> gene along with the expansin  protein-encoding genes exp <dig> and exp <dig>  which are responsive to auxin and contribute to plant growth  <cit> .
when plants were grown on the medium, the root length of ttg2rnai <dig> was much shorter than that of wt rfp , and this difference was statistically significant  as scored in 5–20 days .
similar results were observed on plants grown in pots , and compared to wt rfp, ttg2rnai <dig> was significantly  impaired in the vegetative growth based on the biomass scored as height  and weight  of fresh plants.
consistently, the expression of ntttg <dig>  exp <dig>  and exp <dig> genes was detected at substantial levels in both roots  and leaves  of wt rfp, but little in the ttg2rnai <dig> plant.
these analyses indicate that ntttg <dig> is required for the vegetative growth of tobacco in correlation with the expression of exp <dig> and exp <dig> in the plant.figure  <dig>
the effect of
ntttg2
silencing on tobacco growth.
the ntttg2-silenced tobacco line ttg2rnai <dig> was compared to the control wt rfp plant in terms of the vegetative growth and the expression of ntttg <dig> and exp genes.
root monitoring after 10-day growth on the medium.
plants grown in pots.
floral development stages s1–s <dig> are indicated.
the biomass of plants from .
real-time rt-pcr analyses of the indicated genes expressed in roots of plants from .
real-time rt-pcr analyses of gene expression in leaves of 45-day-old plants grown in pots and equivalent to those in .
in , data shown are mean values ± standard deviation  bas of results from five experimental repeats .
data were analyzed by the anova method along with one-way fisher’s least significant difference  test.
significant differences between data pairs at the corresponding time points are indicated by single asterisks  or double asterisks  on the graphs.
then, we observed the flower and seed development, which has been shown to be regulated by ttg proteins in arabidopsis <cit>  and petunia  <cit> .
we analyzed the effects of ntttg <dig> silencing on the floral development throughout stages s1-s <dig> .
average time to s <dig> was  <dig> and  <dig>  and average time from s <dig> to s <dig> was  <dig> and 9 days in wt rfp and ttg2rnai <dig> plants, respectively.
the effect of ntttg <dig> silencing on flower color was marked.
plants produce anthocyanin, a water-soluble pigment that may make flowers differently colored  <cit> .
wt rfp flowers had visible anthocyanin production since s <dig> and petals appeared red at s <dig> .
floral anthocyanin extracts  and spectrophotometric analyses  indicated that wt rfp flowers started to produce anthocyanin at s <dig> and that abundant amounts of the pigment were detected at s <dig> and s <dig>  by contrast, little anthocyanin was found in ttg2rnai <dig> flowers .figure  <dig>
the effects of
ntttg2
silencing on flower and seed development.
flowers at five development stages  from 120-d-old wt/rfp and 160-d-old ttg2rnai <dig> plants.
flower anthocyanin extracts and spectrophotometric analyses.
the floral anthocyanin content was quantified as relative unit .
real-time rt-pcr analyses of floral transcripts of the ntttg <dig> gene as well as the dfr and ans genes, which are involved in the late stage of the anthocyanin biosynthesis pathway.
growing fruits  from 140-d-old wt/rfp and 170-d-old ttg2rnai <dig>  and mature fruits  from 170-d-old wt/rfp and 210-d-old ttg2rnai <dig> plants.
the number of seeds per mature fruit.
data were given as mean values ± sd bars of results from three  and five  experimental repeats .
significant levels  in differences between corresponding data pairs were analyzed by one-way anova and lsd test.
next, we correlated the variation of anthocyanin production with the expression of the dfr and ans genes involved in late stage of the anthocyanin biosynthesis pathway that mainly comprises nine genes .
the floral expression of dfr and ans was ntttg2-dependent  but that of the other genes was not .
in wt rfp, the expression of ntttg <dig>  dfr, and ans was increased with the floral development, but the three genes were little expressed in ttg2rniai <dig> .
therefore, ntttg <dig> was required for dfr and ans expression with consequent effects on anthocyanin production and flower colorization .
in addition, ntttg <dig> was also required for seed production as ntttg <dig> silencing caused acute seed abortion , and more than 97% seeds were aborted in ttg2rnai <dig> compared to wt rfp .
these analyses indicate that ntttg <dig> functions in flower and seed development of tobacco.
the promoting effect of ntttg <dig> overexpression on tobacco growth and development
to further demonstrate the role of ntttg <dig> in tobacco growth and development, we generated the ntttg2-overexpressing  nc <dig> lines  <cit>  and observed their growth and development characters in comparison with the wt rfp plant.
here, p35s refers to the cauliflower mosaic virus 35s promoter.
in all tested characters, p35s:ttg2:rfp lines were opposite to ttg2rniai <dig>  compared to wt rfp, p35s:ttg2:rfp plants grew better  and had higher levels of exp expression  and biomass .
p35s:ttg2:rfp plants also had better flower colorization , higher levels of ntttg <dig>  dfr, or ans expression from s <dig> through s <dig> , and more anthocyanin production in s <dig> flowers .
moreover, p35s:ttg2:rfp capsules were well developed  with more seeds per fruit .
significant  differences were found between wt rfp and each line of p35s:ttg2:rfp while p35s:ttg2:rfp# <dig> was the best in these developmental characters.
clearly, ntttg <dig> overexpression causes a promoting effect on the vegetative growth and the development of flowers and seeds in tobacco.figure  <dig>
the effects of
ntttg2
overexpression on main characters of tobacco development.
forty-day-old plants.
real-time rt-pcr analyses of exp <dig> and exp <dig> expression in leaves of plants from .
the biomass of plants from .
s <dig> flowers and mature seeds.
s <dig> flower anthocyanin content given as relative unit .
the number of seeds per mature fruit.
quantitative data are presented as mean values ± sd bars of results from five experimental repeats each involving  <dig> plants ,  <dig> flowers , or  <dig> fruits .
different letters on curve and bar graphs indicate significant  differences by one-way anova and lsd test.
transcriptome sequencing and de novo assembly
we sought to identify arf gene candidates that might associate with ntttg2-regulated growth and development in the tobacco n. tabacum variety nc <dig>  we performed de novo assembly of the transcriptome by using the rna-seq technique and a mixture of equal quantities of total rna isolated from leaves of wt rfp, ttg2rnai <dig>  and p35s:ttg2:rfp# <dig>  the mixed rna sample from three biological repeats was used in the library construction and the transcriptome sequencing with the illumina hiseq  <dig> sequencer, followed by de novo assembly with the trinity assembler.
in all,  <dig> , <dig> raw reads were generated, and a total of  <dig> , <dig> clean reads, occupying  <dig> % of total, were obtained after three types of reads  were removed by illumina rna-seq deep-sequencing .
using the high quality reads,  <dig>  transcripts were assembled with an average length of  <dig>  bp, a length variation in 201– <dig>  bp, and  <dig>   transcripts longer than  <dig>  bp.
redundancy rate in  <dig>  transcripts was  <dig> %, and the longest copy of redundant transcripts was regarded as a unigene  <cit> .
in total,  <dig>  unigenes were identified in the transcriptome and their length was found to vary from 201 bp to  <dig>  bp .
the length of  <dig>  ,  <dig>  , and  <dig>   unigenes ranges accordingly in 201– <dig>  501– <dig> ,  <dig> - <dig>  bp while  <dig>   unigenes are longer than  <dig>  bp.figure  <dig>
assessment of the transcriptome assembly quality.
components of raw reads.
the character n in the “containing n” type indicate that nucleotides were not recognized.
different colors represent components.
unigene length distribution.
functional annotation of unigenes
sequences of assembled unigenes were searched against seven different databases  with an e-value threshold of 10- <dig> .
as shown in figure  <dig>  various numbers of unigenes showed significant similarity to the lodged in those databases; similar sequences of  <dig>   unigenes were found in at least one database while  <dig>   unigenes shared similarity to sequences deposited in all databases.
however, sequence orientation of  <dig>   unigenes is still unknown.
based on the e-value distribution of the top hits,  <dig> % of the mapped unigene sequences showed significant homology  with genes deposited in the nr  database .
by searching against the same database,  <dig> % and  <dig> % of the unigenes were found to possess sequence similarities greater than 50% and 75%, respectively .
in regard to organism sources of homologs, approximately  <dig> % of unigenes with nr annotations were matched to plant genes .
while  <dig> %,  <dig> %,  <dig> %,  <dig> %,  <dig> % of the mapped unigene sequences possess significant similarity, respectively with orthologs of ricinus communis, populus trichocarpa, nicotiana tabacum, glycine max, and vitis vinifera .figure  <dig>
distributions of unigenes in seven public databases.
characteristics of similarity search against nr databases.
e-value distribution of blast hits for unigenes with a cutoff e-value of  <dig> e- <dig>   similarity distribution of the top blast hits for unigenes.
organism species distribution of the top blast hits for unigenes in nr database.
the numbers of unigenes are indicated together with percentages placed in parentheses.
functional classification of unigenes
functions of the unigenes were classified with go , an international standardized gene functional classification system that defines genes in three ontologies: “molecular function”, “cellular component”, and “biological process”.
we used the blast2go program  <cit>  to obtain go annotation of the unigenes in the nr database.
then, we used the wego software  <cit>  to classify the unigenes to go function terms.
in total,  <dig>  unigenes  have go annotation, and these unigenes are assigned to  <dig> functional terms .
of these  <dig>  unigenes, the “biological process” occupied the majority , followed by “cellular component”  and “molecular function” .
around the comprehensive range of go categories, “cellular process” , “cell” , “cell part” , “metabolic process” , and “binding”  proteins are in the top five rank and make up the majority of the unigenes .
“cell proliferation”, “extracellular matrix part”, and “nucleotide proteins”  together occupy only  <dig> % of unigenes that have go annotation.
in particular, numbers of unigenes are  <dig> ,  <dig>  ,  <dig>  , and  <dig>   assigned to “growth”, “development process”, “reproduction”, “reproductive process” in the biological process category .
moreover,  <dig>   unigenes fall into the functional term of transcription factor activities in the “molecular function category” .figure  <dig>
go classifications of assembled unigenes.
functions of the unigenes were also classified with the kog/cog  databases that classify predicted unigene products into distinct functional groups  <cit> .
by the kog/cog analysis,  <dig>  of the unigenes were assigned to the  <dig> cog classifications .
some single unigenes were assigned to several cog categories, and this resulted in a record of  <dig>  unigenes in all cog classifications.
in comparison, the category of “general function prediction” contains the greatest number of unigenes , followed by “post-translational modification, protein turnover, and chaperon” , “signal transduction” , “intracellular trafficking and secretion” , “transcription” , and “rna processing and modification” .figure  <dig>
kog/cog classification of unigenes.
metabolic pathway analysis of unigenes
orientation of unigenes in metabolic pathways was analyzed with the kegg  database, which integrates genomic, biochemical, and functional information in various cellular processes  <cit> .
based on blastx searches with an e-value threshold of 10- <dig>   <dig>  of the  <dig>  unigenes were assigned to  <dig> kegg pathways in the kegg database .
those pathways were divided into five branches with numbers/percents of unigenes shown in parentheses: “cellular processes” , “environmental information processing” , “genetic information processing” , “metabolism” , and “organismal systems” .
of the  <dig> kegg pathways, moreover, the pathway of “carbohydrate metabolism” contains homologs of the most unigenes , followed by “translation” , “folding, sorting and degradation” , “signal transduction” , “energy metabolism” , and “amino acid metabolism” , whereas, only no more than  <dig> unigenes were assigned to “signaling molecules and interaction” and “sensory system”.figure  <dig>
kegg classification of unigenes.
capital letters back to the colored bars indicate five main categories: a, cellular processes; b, environmental information processing; c, genetic information processing; d, metabolism; and e, organism systems.
unigenes assigned to auxin responses and the effects of ntttg <dig> on the expression of putative arf genes
browsing of the tobacco transcriptome profile identified  <dig> unigenes assigned to the regulation of auxin responses in plants .
in particular,  <dig>   <dig>   <dig>  and  <dig> unigenes were predicted to be arf, aux/iaa, gh <dig>  and saur gene candidates while additional  <dig>   <dig>  and  <dig> unigenes were categorized as auxin perception, auxin signaling pathway, and response to auxin stimulus, respectively.
this suggests that the tobacco transcriptome is suited for a potential use in further studies to characterize the regulation of auxin responses in the plant.
to examine this notion, we analyzed the effects of ntttg <dig> on the expression of the  <dig> putative arf genes  by testing their expression in wt rfp, ttg2rnai <dig>  and p35s:ttg2:rfp# <dig> plants.
as shown in figure  <dig>  expression levels of  <dig> putative arf genes were similar but the other  <dig> candidates were expressed at different extents in the three genotypes of tobacco.
of the  <dig> differently expressed putative arf genes, two  were downregulated by ntttg <dig> as their transcript abundances were decreased in p35s:ttg2:rfp# <dig> but increased in ttg2rnai <dig>  compared to wt rfp used as a transgenic control plant.
inversely, the other  <dig> putative arf genes  were upregulated by ntttg <dig>  and the expression of these putative genes was enhanced in p35s:ttg2:rfp# <dig> but suppressed in ttg2rnai <dig>  compared to the control plant.
therefore, ntttg <dig> indeed affects the transcript abundance of a relatively small number of arf gene candidates and the effect is either positive or negative depending on particular candidates.figure  <dig>
phylogenic protein families involved in auxin responses and identified in the transcriptome.
the number of genes in the corresponding protein family group  is shown on top of the bar graph.
the “others” group mainly contains auxin-repressed and auxin-induced proteins that do not belong to any of the designated groups.
the effects of ntttg <dig> on the expression of putative
arf
genes.
the effects were determined by analyzing the expression of putative arf genes in wt rfp, p35s:ttg2:rfp# <dig>  and ttg2rnai <dig> plants.
putative arf genes are shown as identification code in the transcriptome inventory.
gene expression levels in the control plant were regarded as  <dig> to qualify relative amounts of the same gene transcripts in the other phenotypes of plant.
data shown are mean values ± sd bars of results from three experimental repeats .
different letters on bar graphs indicate significant  differences by one-way anova and lsd test.
discussion
the repressive effect of the ntttg <dig> gene silencing and the promoting effect of the gene overexpression on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production  suggest that ntttg <dig> is an essential regulator of the growth and development processes in tobacco n. tabacum.
the growth/developmental role of ntttg <dig> associates with its effects on the expression of exp, dfr, and ans genes, which are essentially involved in the plant growth and development pathways  <cit> .
exps have unique “loosening” effects on plant cell walls  <cit>  and promote cell expansion  and plant growth  <cit>  under regulation by phytohormones including auxin  <cit> .
of six exp genes identified in tobacco and hundreds of exps in other plants , exp <dig> and exp <dig> are widely characterized to be required for plant growth .
exp <dig> and exp <dig> expression consistent with the biomass  suggests that both exps contribute to ntttg2-mediated vegetative growth of tobacco.
since the expression of exps is subject to multiple regulations  <cit> , it is not surprising that ntttg <dig> silencing reduces but does not eliminate exp expression.
ntttg <dig> differently affects the expression of genes involved in the anthocyanin biosynthesis pathway .
ntttg <dig> promotes the expression of the dfr and ans genes, which function at the penultimate and antepenultimate steps, respectively in the anthocyanin biosynthesis pathway that mainly involves nine genes  <cit> .
dfr encodes dihydroflavonol 4-reductase that reduces three dihydroflavonols to three leucoanthocyanidins  <cit> , and then two leucoanthocyanidins are converted by ans-encoded anthocyanin synthase to anthocyanidin  <cit> .
the other seven ntttg2-independent genes  encode enzymes with specific actions on single substrates  <cit> .
therefore, ntttg <dig> affects anthocyanin production and flower colorization through affecting the expression of genes critical to the final formation of anthocyanin .
in addition, severe abortions owing to ntttg <dig> silencing and marked promotion of the seed production by the gene overexpression suggest that ntttg <dig> plays a role in seed development .
how ntttg <dig> regulates the expression of dfr and ans, and exps as well, remains to be studied.
to reveal the molecular mechanism that underlies the growth/developmental role of ntttg <dig>  we performed de novo assembly of the tobacco n. tabacum transcriptome to identify ntttg2-affected arf gene candidates in the case that little were known about the regulation of auxin responses in tobacco  <cit> .
the transcriptome was assembled with a high quality based on proportion of clean reads in total raw reads and length sorting of unigenes .
high quality of the transcriptome assembling can be also found in significant homology of unigene sequences with those searched from the public databases .
analyses of the transcriptome have created substantial data on functional components in the plant genome: a total of  <dig>  unigenes,  <dig>  unigenes  with annotated functions, at least  <dig>  unigenes  assigned to plant growth and development, and  <dig> unigenes  related to the regulation of auxin responses  including  <dig> putative arf genes .
in addition,  <dig>   unigenes do not have annotation in any of the databases.
with such substantial genetic components, the n. tabacum transcriptomics inventory should be useful in further studies on a broad perspective of the plant genetics and on physiological or biochemical connections between ttgs and auxin responses in particular.
in support of this notion, we show that ntttg <dig> significantly affects transcript abundances of  <dig> of  <dig> putative arf genes and the effect is either positive or negative depending on particular unigenes .
this indicates that ntttg <dig> may function in association with arf transcription factors in regulating the plant growth and development.
in a recently published draft genome sequence of tobacco n. benthamiana ,  <dig> immunity-associated genes were retrieved from approximately  <dig>  unigenes and this was regarded as evidence for the usefulness of the genome assembly in the study of plant-pathogen interactions  <cit> .
in comparison, the effects of ntttg <dig> on transcript abundances of putative arf genes  suggest that the n. tabacum transcriptome is useful to the study of auxin responses in the plant.
however, the current technique used in de novo transcriptome assembling is unable to obtain full-length sequences of most unigenes.
instead, most unigenes are identified based on partial sequences present in the transcriptome.
in this regard, it is necessary to perform a comprehensive comparison between the  <dig>  unigenes identified in the n. tabacum transcriptome and informative genomic sequence of the plant.
the n. benthamiana genome sequence was established by assembling of  <dig>  sequences with an average length of  <dig>  bp  <cit> .
in the current study, the n. tabacum transcriptome was assembled from  <dig>  transcripts with an average length of  <dig>  bp.
thus, it is rational to speculate that unigenes identified in this study must be much shorter than the actual length of real genes.
due to this reason, further characterizing these unigenes is a great challenge.
it is also a great challenge to characterize the functional relationship between ntttg <dig> and arfs in tobacco.
full-length sequences of the putative arf genes remain unknown except for comp42904_c <dig>  which is 100% identical with the sequence of the arf <dig> gene from the n. tabacum variety nc <dig> .
the predicted arf <dig> protein is characteristic of transcription activators based on glutamine abundance in the middle region of the protein sequence, in contrast to serine abundance in the same region of an arf transcription repressor  <cit> .
unfortunately, information available from the n. tabacum transcriptome is insufficient to show whether the  <dig> arf candidates encode transcription activators or repressors.
cloning of full-length sequences of the arf candidates, analyses of their roles as transcription activators or repressors, and characterization of their functional relationships with ntttg <dig> will be the subject of further studies.
CONCLUSIONS
molecular and genetic analyses performed in this study demonstrate that ntttg <dig> plays an indispensable role in the regulation of growth and development processes in tobacco n. tabacum.
the growth/developmental role of ntttg <dig> associates with its function in regulating the expression of growth-promoting exp genes as well as the dfr and ans genes essentially involved in the anthocyanin biosynthesis pathway.
in the growth/developmental regulation, moreover, ntttg <dig> may have a functional linkage with auxin responses as indicated by the effects of ntttg <dig> silencing and overexpression on the expression of  <dig> putative arf genes identified in the tobacco transcriptome assembled by the de novo assembly method.
substantial data of genetic components derived from analyses of the transcriptome should be useful for studies in the future on the plant genetics in general and on the functional relationship between ntttg <dig> and auxin in regulating growth and development of the plant in particular.

 transparent testa glabra proteins were identified as regulators of trichome and seed development in plants. ttgs are characterized by the presence of the wd motif, which refers to the conserved tryptophan and aspartate dipeptide and the length of approximately amino acid residues in a protein sequence. this motif is present as a series of repeats to form protein interaction domain in a variety of proteins, including all ttgs so far identified except for arabidopsis thaliana atttg which is a wrky transcription factor. as the wd domain has multiple modes for recruiting different substrates, wd40-containing proteins can serve as interchangeable substrate receptors and interact with diverse types of proteins. due to this characteristic, ttgs have the potential of regulating both development and defenses in plants. depending on natures of interacted proteins, ttgs differentiate their functions between development and defense regulations. in arabidopsis, atttg interacts with the bhlh transcription factor gl while recruiting myb gl forming the wd40-bhlh-myb complex that acts to regulate trichome development. previously we demonstrated that wd enabled tobacco ttg to interact with para, an elicitin protein produced by an oomycete pathogen and inducing hypersensitive cell death in the plant. the ntttg1-para interaction was essential for the induction of cell death first in leaf trichomes and then in mesophyll cells. ntttg is an analog of ntttg and both proteins share four wd repeats. recently we showed that ntttg suppressed tobacco resistance to pathogens by indirectly modulating subcellular localization of the npr protein, which is a transcriptional regulator of defense responses to pathogens. ntttg does not interact with npr but is able to sequester npr from the nucleus, thus preventing npr from transcriptional regulation of defense responses. these studies suggest that ttgs regulate specific processes of development or defenses by either directly or indirectly interacting with other proteins involved in the processes. however, whether ntttg plays a role in development regulation was unclear. plant development is subject to a complex regulation of phytohormones with auxin playing a significant role. auxin mediates a stunning array of the development through the functions of auxin response factor proteins in transcriptional regulation of auxin responses. arf transcription factors constitute a phylogenic protein family made of 10– members as identified so far in different plant species. it is accepted that different members of the arf protein family target distinct types of auxin-responsive genes, such as those classified in the small auxin up rna, auxin/indole-acetic acid inducible, and gretchen hagen gene families. however, only a few of auxin-responsive genes have been shown definitely as arf targets and to be regulated directly by specific arfs. in particular, little was known about the regulation of auxin responses in tobacco. in the plant, only four auxin-responsive genes were identified, whereas, at least and members of aux/iaa, saur, and gh gene families were characterized in other plants. in tobacco, moreover, only arf and arf were cloned with full length cdna sequences while additional eleven arf homologs were identified as expressed sequence tags in contrast to the 10– members of the arf protein family already identified in other plants. therefore, it is rational to conjecture that tobacco may possess more arfs and auxin-responsive genes than the currently known numbers. in addition, there is as yet no study to characterize functions of arfs in tobacco. this study was attempted to elucidate the role of ntttg in the growth and development of tobacco and to identify arf gene candidates associated with ntttg2-regulated growth and development. we show that ntttg is required for the vegetative growth and the development of flowers and seeds. we performed de novo assembly of the tobacco n. tabacum transcriptome and analyzed functional components in the transcriptome. by using transcriptome information, we identified the unigenes assigned to auxin responses and representing putative arf genes in particular. we present evidence that ntttg affects the expression of putative arf genes in the plant.  the repressive effect of ntttg silencing on the vegetative growth and the development of flowers and seeds in tobacco to elucidate the roles of ntttg in the growth and development of tobacco n. tabacum, we generated the ntttg2-silencing line ttg2rnai under background of the n. tabacum variety nc and observed its growth and development in contrast to the wild-type phenotype. the transgenic nc line wt rfp was used as a control since it highly resembled wt in the development process and biological productivity. rfp is a red-fluorescent protein-encoding gene used in detecting subcellular localization of the protein but is not related to this study. molecular and genetic characterizations of transgenic plants have been documented in a recently published article. in this study, we investigated the vegetative growth of wt rfp and ttg2rnai plants on murashige and skoog agar medium and in pots. we also analyzed the expression of the ntttg gene along with the expansin protein-encoding genes exp and exp which are responsive to auxin and contribute to plant growth. when plants were grown on the medium, the root length of ttg2rnai was much shorter than that of wt rfp, and this difference was statistically significant as scored in 5–20 days. similar results were observed on plants grown in pots, and compared to wt rfp, ttg2rnai was significantly impaired in the vegetative growth based on the biomass scored as height and weight of fresh plants. consistently, the expression of ntttg exp and exp genes was detected at substantial levels in both roots and leaves of wt rfp, but little in the ttg2rnai plant. these analyses indicate that ntttg is required for the vegetative growth of tobacco in correlation with the expression of exp and exp in the plant.figure the effect of ntttg2 silencing on tobacco growth. the ntttg2-silenced tobacco line ttg2rnai was compared to the control wt rfp plant in terms of the vegetative growth and the expression of ntttg and exp genes. root monitoring after 10-day growth on the medium. plants grown in pots. floral development stages s1–s are indicated. the biomass of plants from. real-time rt-pcr analyses of the indicated genes expressed in roots of plants from. real-time rt-pcr analyses of gene expression in leaves of 45-day-old plants grown in pots and equivalent to those in. in, data shown are mean values ± standard deviation bas of results from five experimental repeats. data were analyzed by the anova method along with one-way fisher’s least significant difference test. significant differences between data pairs at the corresponding time points are indicated by single asterisks or double asterisks on the graphs. then, we observed the flower and seed development, which has been shown to be regulated by ttg proteins in arabidopsis and petunia. we analyzed the effects of ntttg silencing on the floral development throughout stages s1-s. average time to s was and and average time from s to s was and 9 days in wt rfp and ttg2rnai plants, respectively. the effect of ntttg silencing on flower color was marked. plants produce anthocyanin, a water-soluble pigment that may make flowers differently colored. wt rfp flowers had visible anthocyanin production since s and petals appeared red at s. floral anthocyanin extracts and spectrophotometric analyses indicated that wt rfp flowers started to produce anthocyanin at s and that abundant amounts of the pigment were detected at s and s by contrast, little anthocyanin was found in ttg2rnai flowers.figure the effects of ntttg2 silencing on flower and seed development. flowers at five development stages from 120-d-old wt/rfp and 160-d-old ttg2rnai plants. flower anthocyanin extracts and spectrophotometric analyses. the floral anthocyanin content was quantified as relative unit. real-time rt-pcr analyses of floral transcripts of the ntttg gene as well as the dfr and ans genes, which are involved in the late stage of the anthocyanin biosynthesis pathway. growing fruits from 140-d-old wt/rfp and 170-d-old ttg2rnai and mature fruits from 170-d-old wt/rfp and 210-d-old ttg2rnai plants. the number of seeds per mature fruit. data were given as mean values ± sd bars of results from three and five experimental repeats. significant levels in differences between corresponding data pairs were analyzed by one-way anova and lsd test. next, we correlated the variation of anthocyanin production with the expression of the dfr and ans genes involved in late stage of the anthocyanin biosynthesis pathway that mainly comprises nine genes. the floral expression of dfr and ans was ntttg2-dependent but that of the other genes was not. in wt rfp, the expression of ntttg dfr, and ans was increased with the floral development, but the three genes were little expressed in ttg2rniai. therefore, ntttg was required for dfr and ans expression with consequent effects on anthocyanin production and flower colorization. in addition, ntttg was also required for seed production as ntttg silencing caused acute seed abortion, and more than 97% seeds were aborted in ttg2rnai compared to wt rfp. these analyses indicate that ntttg functions in flower and seed development of tobacco. the promoting effect of ntttg overexpression on tobacco growth and development to further demonstrate the role of ntttg in tobacco growth and development, we generated the ntttg2-overexpressing nc lines and observed their growth and development characters in comparison with the wt rfp plant. here, p35s refers to the cauliflower mosaic virus 35s promoter. in all tested characters, p35s:ttg2:rfp lines were opposite to ttg2rniai compared to wt rfp, p35s:ttg2:rfp plants grew better and had higher levels of exp expression and biomass. p35s:ttg2:rfp plants also had better flower colorization, higher levels of ntttg dfr, or ans expression from s through s, and more anthocyanin production in s flowers. moreover, p35s:ttg2:rfp capsules were well developed with more seeds per fruit. significant differences were found between wt rfp and each line of p35s:ttg2:rfp while p35s:ttg2:rfp# was the best in these developmental characters. clearly, ntttg overexpression causes a promoting effect on the vegetative growth and the development of flowers and seeds in tobacco.figure the effects of ntttg2 overexpression on main characters of tobacco development. forty-day-old plants. real-time rt-pcr analyses of exp and exp expression in leaves of plants from. the biomass of plants from. s flowers and mature seeds. s flower anthocyanin content given as relative unit. the number of seeds per mature fruit. quantitative data are presented as mean values ± sd bars of results from five experimental repeats each involving plants, flowers, or fruits. different letters on curve and bar graphs indicate significant differences by one-way anova and lsd test. transcriptome sequencing and de novo assembly we sought to identify arf gene candidates that might associate with ntttg2-regulated growth and development in the tobacco n. tabacum variety nc we performed de novo assembly of the transcriptome by using the rna-seq technique and a mixture of equal quantities of total rna isolated from leaves of wt rfp, ttg2rnai and p35s:ttg2:rfp# the mixed rna sample from three biological repeats was used in the library construction and the transcriptome sequencing with the illumina hiseq sequencer, followed by de novo assembly with the trinity assembler. in all,, raw reads were generated, and a total of, clean reads, occupying % of total, were obtained after three types of reads were removed by illumina rna-seq deep-sequencing. using the high quality reads, transcripts were assembled with an average length of  bp, a length variation in 201–  bp, and transcripts longer than  bp. redundancy rate in transcripts was %, and the longest copy of redundant transcripts was regarded as a unigene. in total, unigenes were identified in the transcriptome and their length was found to vary from 201 bp to  bp. the length of,, and unigenes ranges accordingly in 201– 501–, -  bp while unigenes are longer than  bp.figure assessment of the transcriptome assembly quality. components of raw reads. the character n in the “containing n” type indicate that nucleotides were not recognized. different colors represent components. unigene length distribution. functional annotation of unigenes sequences of assembled unigenes were searched against seven different databases with an e-value threshold of 10-. as shown in figure  various numbers of unigenes showed significant similarity to the lodged in those databases; similar sequences of unigenes were found in at least one database while unigenes shared similarity to sequences deposited in all databases. however, sequence orientation of unigenes is still unknown. based on the e-value distribution of the top hits, % of the mapped unigene sequences showed significant homology with genes deposited in the nr database. by searching against the same database, % and % of the unigenes were found to possess sequence similarities greater than 50% and 75%, respectively. in regard to organism sources of homologs, approximately % of unigenes with nr annotations were matched to plant genes. while %, %, %, %, % of the mapped unigene sequences possess significant similarity, respectively with orthologs of ricinus communis, populus trichocarpa, nicotiana tabacum, glycine max, and vitis vinifera.figure distributions of unigenes in seven public databases. characteristics of similarity search against nr databases. e-value distribution of blast hits for unigenes with a cutoff e-value of e- similarity distribution of the top blast hits for unigenes. organism species distribution of the top blast hits for unigenes in nr database. the numbers of unigenes are indicated together with percentages placed in parentheses. functional classification of unigenes functions of the unigenes were classified with go, an international standardized gene functional classification system that defines genes in three ontologies: “molecular function”, “cellular component”, and “biological process”. we used the blast2go program to obtain go annotation of the unigenes in the nr database. then, we used the wego software to classify the unigenes to go function terms. in total, unigenes have go annotation, and these unigenes are assigned to functional terms. of these unigenes, the “biological process” occupied the majority, followed by “cellular component” and “molecular function”. around the comprehensive range of go categories, “cellular process”, “cell”, “cell part”, “metabolic process”, and “binding” proteins are in the top five rank and make up the majority of the unigenes. “cell proliferation”, “extracellular matrix part”, and “nucleotide proteins” together occupy only % of unigenes that have go annotation. in particular, numbers of unigenes are,,, and assigned to “growth”, “development process”, “reproduction”, “reproductive process” in the biological process category. moreover, unigenes fall into the functional term of transcription factor activities in the “molecular function category”.figure go classifications of assembled unigenes. functions of the unigenes were also classified with the kog/cog databases that classify predicted unigene products into distinct functional groups. by the kog/cog analysis, of the unigenes were assigned to the cog classifications. some single unigenes were assigned to several cog categories, and this resulted in a record of unigenes in all cog classifications. in comparison, the category of “general function prediction” contains the greatest number of unigenes, followed by “post-translational modification, protein turnover, and chaperon”, “signal transduction”, “intracellular trafficking and secretion”, “transcription”, and “rna processing and modification”.figure kog/cog classification of unigenes. metabolic pathway analysis of unigenes orientation of unigenes in metabolic pathways was analyzed with the kegg database, which integrates genomic, biochemical, and functional information in various cellular processes. based on blastx searches with an e-value threshold of 10- of the unigenes were assigned to kegg pathways in the kegg database. those pathways were divided into five branches with numbers/percents of unigenes shown in parentheses: “cellular processes”, “environmental information processing”, “genetic information processing”, “metabolism”, and “organismal systems”. of the kegg pathways, moreover, the pathway of “carbohydrate metabolism” contains homologs of the most unigenes, followed by “translation”, “folding, sorting and degradation”, “signal transduction”, “energy metabolism”, and “amino acid metabolism”, whereas, only no more than unigenes were assigned to “signaling molecules and interaction” and “sensory system”.figure kegg classification of unigenes. capital letters back to the colored bars indicate five main categories: a, cellular processes; b, environmental information processing; c, genetic information processing; d, metabolism; and e, organism systems. unigenes assigned to auxin responses and the effects of ntttg on the expression of putative arf genes browsing of the tobacco transcriptome profile identified unigenes assigned to the regulation of auxin responses in plants. in particular, and unigenes were predicted to be arf, aux/iaa, gh and saur gene candidates while additional and unigenes were categorized as auxin perception, auxin signaling pathway, and response to auxin stimulus, respectively. this suggests that the tobacco transcriptome is suited for a potential use in further studies to characterize the regulation of auxin responses in the plant. to examine this notion, we analyzed the effects of ntttg on the expression of the putative arf genes by testing their expression in wt rfp, ttg2rnai and p35s:ttg2:rfp# plants. as shown in figure  expression levels of putative arf genes were similar but the other candidates were expressed at different extents in the three genotypes of tobacco. of the differently expressed putative arf genes, two were downregulated by ntttg as their transcript abundances were decreased in p35s:ttg2:rfp# but increased in ttg2rnai compared to wt rfp used as a transgenic control plant. inversely, the other putative arf genes were upregulated by ntttg and the expression of these putative genes was enhanced in p35s:ttg2:rfp# but suppressed in ttg2rnai compared to the control plant. therefore, ntttg indeed affects the transcript abundance of a relatively small number of arf gene candidates and the effect is either positive or negative depending on particular candidates.figure phylogenic protein families involved in auxin responses and identified in the transcriptome. the number of genes in the corresponding protein family group is shown on top of the bar graph. the “others” group mainly contains auxin-repressed and auxin-induced proteins that do not belong to any of the designated groups. the effects of ntttg on the expression of putative arf genes. the effects were determined by analyzing the expression of putative arf genes in wt rfp, p35s:ttg2:rfp# and ttg2rnai plants. putative arf genes are shown as identification code in the transcriptome inventory. gene expression levels in the control plant were regarded as to qualify relative amounts of the same gene transcripts in the other phenotypes of plant. data shown are mean values ± sd bars of results from three experimental repeats. different letters on bar graphs indicate significant differences by one-way anova and lsd test. discussion the repressive effect of the ntttg gene silencing and the promoting effect of the gene overexpression on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production suggest that ntttg is an essential regulator of the growth and development processes in tobacco n. tabacum. the growth/developmental role of ntttg associates with its effects on the expression of exp, dfr, and ans genes, which are essentially involved in the plant growth and development pathways. exps have unique “loosening” effects on plant cell walls and promote cell expansion and plant growth under regulation by phytohormones including auxin. of six exp genes identified in tobacco and hundreds of exps in other plants, exp and exp are widely characterized to be required for plant growth. exp and exp expression consistent with the biomass suggests that both exps contribute to ntttg2-mediated vegetative growth of tobacco. since the expression of exps is subject to multiple regulations, it is not surprising that ntttg silencing reduces but does not eliminate exp expression. ntttg differently affects the expression of genes involved in the anthocyanin biosynthesis pathway. ntttg promotes the expression of the dfr and ans genes, which function at the penultimate and antepenultimate steps, respectively in the anthocyanin biosynthesis pathway that mainly involves nine genes. dfr encodes dihydroflavonol 4-reductase that reduces three dihydroflavonols to three leucoanthocyanidins, and then two leucoanthocyanidins are converted by ans-encoded anthocyanin synthase to anthocyanidin. the other seven ntttg2-independent genes encode enzymes with specific actions on single substrates. therefore, ntttg affects anthocyanin production and flower colorization through affecting the expression of genes critical to the final formation of anthocyanin. in addition, severe abortions owing to ntttg silencing and marked promotion of the seed production by the gene overexpression suggest that ntttg plays a role in seed development. how ntttg regulates the expression of dfr and ans, and exps as well, remains to be studied. to reveal the molecular mechanism that underlies the growth/developmental role of ntttg we performed de novo assembly of the tobacco n. tabacum transcriptome to identify ntttg2-affected arf gene candidates in the case that little were known about the regulation of auxin responses in tobacco. the transcriptome was assembled with a high quality based on proportion of clean reads in total raw reads and length sorting of unigenes. high quality of the transcriptome assembling can be also found in significant homology of unigene sequences with those searched from the public databases. analyses of the transcriptome have created substantial data on functional components in the plant genome: a total of unigenes, unigenes with annotated functions, at least unigenes assigned to plant growth and development, and unigenes related to the regulation of auxin responses including putative arf genes. in addition, unigenes do not have annotation in any of the databases. with such substantial genetic components, the n. tabacum transcriptomics inventory should be useful in further studies on a broad perspective of the plant genetics and on physiological or biochemical connections between ttgs and auxin responses in particular. in support of this notion, we show that ntttg significantly affects transcript abundances of of putative arf genes and the effect is either positive or negative depending on particular unigenes. this indicates that ntttg may function in association with arf transcription factors in regulating the plant growth and development. in a recently published draft genome sequence of tobacco n. benthamiana, immunity-associated genes were retrieved from approximately unigenes and this was regarded as evidence for the usefulness of the genome assembly in the study of plant-pathogen interactions. in comparison, the effects of ntttg on transcript abundances of putative arf genes suggest that the n. tabacum transcriptome is useful to the study of auxin responses in the plant. however, the current technique used in de novo transcriptome assembling is unable to obtain full-length sequences of most unigenes. instead, most unigenes are identified based on partial sequences present in the transcriptome. in this regard, it is necessary to perform a comprehensive comparison between the unigenes identified in the n. tabacum transcriptome and informative genomic sequence of the plant. the n. benthamiana genome sequence was established by assembling of sequences with an average length of  bp. in the current study, the n. tabacum transcriptome was assembled from transcripts with an average length of  bp. thus, it is rational to speculate that unigenes identified in this study must be much shorter than the actual length of real genes. due to this reason, further characterizing these unigenes is a great challenge. it is also a great challenge to characterize the functional relationship between ntttg and arfs in tobacco. full-length sequences of the putative arf genes remain unknown except for comp42904_c which is 100% identical with the sequence of the arf gene from the n. tabacum variety nc. the predicted arf protein is characteristic of transcription activators based on glutamine abundance in the middle region of the protein sequence, in contrast to serine abundance in the same region of an arf transcription repressor. unfortunately, information available from the n. tabacum transcriptome is insufficient to show whether the arf candidates encode transcription activators or repressors. cloning of full-length sequences of the arf candidates, analyses of their roles as transcription activators or repressors, and characterization of their functional relationships with ntttg will be the subject of further studies.  molecular and genetic analyses performed in this study demonstrate that ntttg plays an indispensable role in the regulation of growth and development processes in tobacco n. tabacum. the growth/developmental role of ntttg associates with its function in regulating the expression of growth-promoting exp genes as well as the dfr and ans genes essentially involved in the anthocyanin biosynthesis pathway. in the growth/developmental regulation, moreover, ntttg may have a functional linkage with auxin responses as indicated by the effects of ntttg silencing and overexpression on the expression of putative arf genes identified in the tobacco transcriptome assembled by the de novo assembly method. substantial data of genetic components derived from analyses of the transcriptome should be useful for studies in the future on the plant genetics in general and on the functional relationship between ntttg and auxin in regulating growth and development of the plant in particular. 
to find the functional linkage between ttg and auxin in the regulation of plant development, we performed de novo assembly of the tobacco transcriptome to identify candidates of ntttg2-regulated arf genes. the growth/developmental function of ntttg associates with differential expression of putative arf genes identified by de novo assembly of the tobacco transcriptome. the transcriptome also contains unigenes related to auxin responses, including putative arf genes. the effects of the altered ntttg expression on expression levels of putative arf genes identified in the transcriptome suggest that ntttg functions in relation to arf transcription factors. the role of ntttg in tobacco growth and development was studied by analyzing the biological effects of gene silencing and overexpression. by contrast, the plant growth and development processes are promoted by ntttg overexpression. the phytohormone auxin mediates a stunning array of plant development through the functions of auxin response factors, which belong to transcription factors and are present as a protein family comprising 10– members so far identified in different plant species. the biological effects of the ntttg gene silencing and overexpression suggest that ntttg is an essential regulator of growth and development in tobacco. the ntttg gene silencing causes repressive effects on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production. the transcriptome contains a total of unigenes, including unigenes with annotated functions and at least unigenes assigned to plant growth and development. based on quantitative analyses, the expression of the putative genes is either promoted or inhibited by ntttg plant development is also subject to regulation by transparent testa glabra proteins, such as ntttg that we recently characterized in tobacco nicotiana tabacum.
 the phytohormone auxin mediates a stunning array of plant development through the functions of auxin response factors, which belong to transcription factors and are present as a protein family comprising 10– members so far identified in different plant species. plant development is also subject to regulation by transparent testa glabra proteins, such as ntttg that we recently characterized in tobacco nicotiana tabacum. to find the functional linkage between ttg and auxin in the regulation of plant development, we performed de novo assembly of the tobacco transcriptome to identify candidates of ntttg2-regulated arf genes.  the role of ntttg in tobacco growth and development was studied by analyzing the biological effects of gene silencing and overexpression. the ntttg gene silencing causes repressive effects on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production. by contrast, the plant growth and development processes are promoted by ntttg overexpression. the growth/developmental function of ntttg associates with differential expression of putative arf genes identified by de novo assembly of the tobacco transcriptome. the transcriptome contains a total of unigenes, including unigenes with annotated functions and at least unigenes assigned to plant growth and development. the transcriptome also contains unigenes related to auxin responses, including putative arf genes. based on quantitative analyses, the expression of the putative genes is either promoted or inhibited by ntttg  the biological effects of the ntttg gene silencing and overexpression suggest that ntttg is an essential regulator of growth and development in tobacco. the effects of the altered ntttg expression on expression levels of putative arf genes identified in the transcriptome suggest that ntttg functions in relation to arf transcription factors. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© biomed central ltd 2013 