BACKGROUND
small rna silencing is an essential mechanism in gene regulation in eukaryotes.
there are two main categories of small rnas: micrornas  and small interference rnas , which are generated from single-stranded, self-complementary rna transcripts and double-stranded rnas , respectively.
the primary transcript of a mir gene is called pri-mirna, which is further processed into the stem-loop precursor mirna  by dicer like  <dig> .
while the guide strands of the mirna duplexes are incorporated into argonaute  <dig> of the rna-induced silencing complex , the passenger strands called mirna star  are mostly degraded   <cit> .
the sources of dsrnas that trigger sirnas biogenesis could be exogenous  or endogenous.
plant evolved several classes of endogenous sirnas including tasirnas, natsirnas and cis-acting sirnas .
in plants, tasirnas are generated by a pathway different from that of mirnas .
the genomic loci encoding tasirnas are known as tas genes and are transcribed by polii.
the generation of tasirna is initiated by mirna-mediated cleavage of long non-coding transcripts of tas genes.
eight tas loci from four families  are identified in arabidopsis genome  <cit> .
there are three loci in tas <dig> family, tas1a , tas1b  and tas1c .
both tas <dig> and tas <dig>  transcripts are cleaved by mir <dig> and associated with ago <dig> to generate sirnas, which mainly target pentatricopeptide repeat-containing  mrnas  <cit> .
there are three tas <dig> loci in arabidopsis thaliana, tas3a , tas3b  and tas3c .
mir <dig> guides cleavage of these transcripts with ago <dig> to generate sirnas which target mrnas of auxin responsive factors  family   <cit> .
tas <dig> transcript is initiated by mir <dig> in association with ago <dig> to generate tasirnas and their targets are myb transcription factors  <cit> .
the cleaved rnas from the eight loci are bounded by suppressor of gene silencing  <dig> and copied into dsrnas by rna-dependent rna polymerase  <dig> .
the dsrnas are cleaved in multiple rounds by dcl <dig> from the end defined by mirna-mediated cleavage such that the tasirnas are in 21-nucleotide  register from the cleavage site.
the tasirnas are loaded into ago <dig> complex to initiate tasirna guided mrna degradation  <cit> .
another class of sirnas is nat-sirnas, which could be derived from rnas transcribed from opposite strands of the same loci   <cit>  or by transcripts from different loci .
there are  <dig>  and  <dig>  potential cis- and trans- natural antisense transcripts , respectively in arabidopsis <cit> .
the production of nat-sirnas are is dependent on rdr <dig> and dcl <dig>  or dcl <dig> .
overexpression of atpap <dig> in a. thaliana speeds up plant growth.
the oe lines flower early and grow faster than the wild type  plants.
the seed yield and silique numbers of oe lines are also more than the control lines  <cit> .
atpap <dig> was shown to be dually targeted to chloroplasts and mitochondria  <cit> .
metabolomics studies showed that some sugars , tca metabolites  and certain amino acids  are significantly higher in the oe lines than in the wt  <cit> .
the concentrations of atp and adp are also higher in the oe lines  <cit> .
all of these phenotypes pointed to a dramatic shift of metabolism in the overexpression lines  <cit> .
besides, overexpression of atpap <dig> in another member of brassicaceae  can also generate fast growing and higher seed-yield transgenic plants  <cit> .
microarray data showed that thousands of transcripts were significantly altered  in the oe lines  <cit> , including key genes involved in carbon flow, k uptake and nitrogen assimilation.
in addition, the transcription of many genes involved in mirnas and sirnas biogenesis was altered .
the transcript abundance of key genes involved in mirna biogenesis, including dcl <dig> and hyl <dig>  significantly increased  in both oe lines, whereas sdn <dig>  responsible for mirna degradation, had transcript abundance decreased in both oe lines .
the transcript abundance of essential genes involved in tasirna biogenesis, including sgs <dig>  ago <dig> and ago <dig>  also significantly increased  in both oe lines.
to examine the small rnas profiles in the fast-growing lines, we constructed eight srna libraries from both leaves and roots of wild type , pap <dig> , and two independent overexpression lines .
this study provides information on the impacts of high energy status  on small rna profiles in arabidopsis.
pap2
, oe <dig> and oe21
pap2
*significantly  changed in both oe lines.
fc: fold change.
the following genes were not significantly  different between the wt and oe lines:
ago <dig>  ago <dig>  ago <dig>  dcl <dig>  dcl <dig>  hen  <dig>  hen <dig>  hen <dig>  hasty, nrpd1a, rdr <dig>  rdr <dig> , sde <dig> and sde <dig>
RESULTS
small rnas length distribution and annotation
eight small rna libraries were generated from the leaves and the roots of 20-day-old arabidopsis.
after removal of adaptors and low quality tags, the number of cleans reads were calculated and mapped to the a. thaliana genome.
in total,  <dig> to  <dig> million raw reads were generated from each library, and approximately 95% of the reads remained for further research after clearing the adaptors sequences.
approximately  <dig> to  <dig> million sequence reads  corresponding to  <dig>  to  <dig>  million unique sequence signatures could be mapped onto the genome .
the length of the clean reads ranged from  <dig> to  <dig> nucleotides.
the size distribution of small rna sequences in various leaf  and root  samples were reported in additional file  <dig>  similar distributions were observed in oe, pap <dig> and wild type leaf libraries.
consistent with the earlier reports  <cit> , two major peaks appeared at  <dig> and  <dig> nucleotides in length in the total sequence reads .
in the root libraries, an additional peak at  <dig> nucleotides was observed.
these 19-nucletide sequences are mainly originated from trnas, and they are cleaved from the 5′ end of gly-trnatcc which represented over 80% of trna-derived small rnas in the roots  <cit> .
interestingly, the total reads of these 19-nucletide sequences are relatively more  in both oe lines than in wt  and pap <dig>  .
clean reads from the eight libraries were mapped and classified according to the annotation of the genome.
a significant higher proportion of small rnas were mapped to trna genes in the root libraries  than in the leaf libraries .
for the repeat small rnas, both unique and total reads are less in oe leaf and root samples.
we observed that the total reads of srna antisense to exons  are higher in leaves of the oe lines, while there are smaller number of unique reads ; these indicate that the srna antisense to exons are more concentrated to their targets in the leaves of oe lines.
identification of known mirnas
it has been reported that high throughput sequencing can be an alternative to estimate the expression profiles of mirna genes  <cit> .
this method allows us to distinguish the transcript abundance of the same mirna gene between different lines.
small rnas were mapped to the mirna precursor/mature mirna species in mirbase <dig>  .
expression profiles of all known mirnas of the eight libraries were listed including  <dig> mirnas .
scatter plot  showed the differential expression of mirna between different lines.
the mirnas expression patterns between the two oe lines are indifferent, while comparing to wt as controls, there are significant changes.
table  <dig> shows the mirnas with significant differential expression between the oe lines and the wt.
all the known mirnas were calculated by fold change log <dig>  > <dig> or < - <dig> and p-value < <dig> .
the mirnas significantly changed in the oe lines mainly target to the mrnas of several protein families including spl family, hap2-like transcription factors, apetala 2-like transcription factors, tcp transcription factors, tas family, laccases, cation/hydrogen exchangers and jacalin lectins.
these mirna expression data was cross-examined with the leaf microarray data.
increases in some mirnas abundance correlates with the decreases in mrna transcript abundance, including ath-mir172a,b, ath-mir172c, ath-mir319a,b and ath-mir <dig>  the targets are apetala2-like transcription factors , tcp transcription factors and jacalin lectins, respectively.
the abundance of mir <dig>  well known to be responsible for initiating tasirna biogenesis for tas1a, tas1b, tas1c and tas <dig>  significantly increased in the leaves of both oe lines.
in contrast, the abundance of mir <dig>  a member of the mir <dig> family  <cit> , significantly decreased in the leaves of both oe lines.
pap2
pap2
numbers in bold mean significant changes in oe versus wt, p-value < <dig>  and fold change log <dig>  > <dig> or fold change log <dig>  < - <dig>  all reads were normalized.
the reads cut off was  <dig>  all the targets were predicted from website of mayer’s lab .
analysis of novel mirnas candidates and their targets
novel mirnas and their target genes were predicted by the mireap software.
target genes are predicted based on the rules suggested by allen et al.
<cit>  and schwab  <cit> .
secondary structures were predicted and analyzed for stable stem-loop hairpins .
the detail of novel mir genes including location, minus free energy , sequence and structures are listed in additional file  <dig>  according to the criteria described in methods, seven novel mirnas with counts >  <dig> were identified in leaf libraries, of which two were also identified in root libraries .
for leaf/root_mirna0001rna_5p, it was predicted to target to chromomethylase <dig> .
and for leaf_mirna0002_5p and root_mirna0002_3p, only found in wt/pap <dig>  were predicted to target retrotransposon orf- <dig> protein.
leaf_mirna0003_3p and leaf_mirna008_5p, which had relatively high reads, were only found in pap <dig>  and wt , respectively and their targets were six f-box family proteins.
leaf_mirna0005_5p and leaf_mirna0006_3p were predicted to target pentatricopeptide repeat  family proteins , both are proteins located in mitochondria.
these two mirnas were either not found  or the counts were too low  in the leaves of wt and pap <dig> line and therefore not detected in previous studies.
trans-acting small interference srnas analysis
the mapping of all the clean reads to the eight tasirnas loci, 21-nt tasirna reads were normalized and compared between samples.
there were significant changes in the tasirna profiles in the leaves of both oe lines .
as stated above, the counts of mir <dig> were significantly higher in the leaves of both oe lines.
mir <dig> was known to induce cleavage of tas <dig> and tas <dig> transcripts and initiate tasirnas production from these loci .
our data shows that the amounts of tasirnas generated from tas1a, b, c and tas <dig> were significantly increased in the leaves of the oe lines.
phase register and count distribution of tas1a, tas1b, tas1c and tas <dig> sites cleaved by mir <dig> in leaf were presented .
the phase distribution pattern of tas1b was significantly altered between oe and wt lines .
the predicted targets of many of these tasirna are mrnas of ppr and tpr genes.
in the four leaf samples,  <dig> individual 21-nt srna sequences with reads > <dig> could be mapped to the four tas <dig> and tas <dig> loci, of which  <dig> srna sequences were found to be significantly differentially expressed between the wt and both oe lines .
the same srna sequences could be generated from more than one locus, and multiple srnas are predicted to target to the same target.
a network of mir173-tasirnas-ppr/tpr was generated to show their relationship .
all the potential ppr/tpr genes that appeared in the network were listed in additional file  <dig>  for tas <dig>  the abundance of tasirna tas4-sir <dig>   strongly decreased in both oe lines  compared to the wt .
in contrast, there are no significant changes in the tasirnas generated from the three tas <dig> loci .
natural-antisense-transcript derived small interference rnas
out of  <dig>  potential cis-nats predicted from arabidopsis tair  <dig> database, we could only map our srnas to  <dig> cis-nats; only  <dig>  and  <dig>  pairs were significantly altered in the leaves and roots of both oe lines .
only  <dig> cis-nats increased in abundance in the leaves of the oe lines, some have significant fold changes.
the genes in the gene pairs include  <dig> mirnas , tas1c, one ppr gene, two genes involved in auxin response, a histone h2a gene and a phosphoribosylanthranilate isomerase gene.
the srna counts for the latter two gene pairs increased by 10- and 60-fold in leaves, respectively.
some cis-nats strongly decreased in abundance in the leaves of oe lines.
the genes in the gene pairs include genes of lhcb <dig> , lhcb <dig>  a protein kinase apk2b, 2 f-box proteins and a udp-3-o- n-acetylglucosamine deacetylase.
the srna mapped to the latter gene pair dropped ~100-fold in both leaves and roots.
out of  <dig>  possible trans-nats pairs, only  <dig>  and  <dig>  pairs were significantly altered in the leaves and roots of both oe lines, respectively .
most of the genes involved are transponsable elements , pseudogenes  and genes for unknown proteins .
validation of sirnas and candidate genes by qrt-pcr
to validate the srna sequencing data, the abundance of selected mirnas  and 21-nt tasi-rnas mapped to cds of tasib , tas1c , tas <dig>  and tas4-sir <dig> in different lines were compared by qrt-pcr.
all the results of selected small rnas were consistent between qrt-pcr and sequencing.
the microarray data has been validated by qrt-pcr in our previous study  <cit>  and the transcript abundance of selected genes, including sgs <dig>  at1g <dig> , were also well validated by qrt-pcr in this study .
discussion
srna biogenesis is one of the regulatory mechanisms in organism.
in this study, the srna profiles of high energy plants were compared with that of wt plants under soil-grown condition.
the size and growth stages of the plants were identical so that variations due to developmental stages and morphology were minimized.
many mirnas and sirnas were activated by various biotic and abiotic stresses and subsequently modulated the mrna stability of their target genes, so that the plants can cope with the stresses accordingly  <cit> .
our data shows that very few mirnas were significantly altered in the oe lines under the experimental conditions.
out of  <dig> known mirnas in arabidopsis, only  <dig> and  <dig> mirnas significantly increased or decreased in abundance in the leaves of oe lines.
very few mirnas were altered in the roots, only  <dig> and  <dig> mirnas significantly increased or decreased in abundance, respectively.
some mirnas were shown to control plant development  <cit> .
overexpression of mir <dig> and mir <dig> resulted in late-flowering  <cit> , and overexpression of mir <dig> resulted in increased lateral rooting  <cit> .
however, the abundance of these mirnas was unaltered in the oe lines.
only the abundance of mir <dig> and mir <dig> were significantly higher in the leaves of the oe lines .
the targets of mir <dig> and mir <dig> are ap <dig> and tcp, transcription factors, respectively.
both are involved in leaf development, floral organ identity and flowering time  <cit> .
overexpression of mir <dig> in arabidopsis and potato can promote flowering development and tuberization respectively  <cit> .
the higher expression of mir <dig> in the leaves of oe lines correlates with the earlier bolting and flowering phenotypes of the oe lines  <cit> .
for mirnas that are induced by nutritional stresses, such as phosphate deficiency  copper deficiency , and sulfate deficiency , their abundance were low in all lines and were not significantly changed in our oe lines  <cit> .
this is reasonable as the plants were grown in soil with adequate supply of nutrients.
similarly, mirnas responsive to bacterial  and viral infections  were not altered in the oe lines  <cit> .
the abundance of some mirna  induced by cold stresses increased in the oe lines but many other mirnas  induced by cold were unaltered  <cit> .
accumulation of sucrose in the cytosol is a key protection mechanism for cold tolerance.
since the oe lines contain higher sucrose contents  <cit> , mir <dig> and mir <dig> might be induced indirectly by cold-induced sucrose accumulation.
currently, eight tas genes of tasirnas have been identified in the arabidopsis genome.
the transcribed mrnas from these loci require mirna-induced cleavage to generate functional tasirnas, which in turn induce the cleavage of the target mrnas.
for example, the tas1a/b/c and tas <dig> tasirna families are induced by mir <dig>  which then down-regulate mrnas of various ppr and tpr genes  <cit> .our data shows that the mir173-tasirnas-ppr/tpr network had substantial changes in the leaves of both oe lines .
a tas gene prediction algorithm predicted seven tas loci in a. thaliana  using col- <dig> mpss small rna data  <cit> , which include tas1a/b/c, tas <dig>  and  <dig> ppr genes .
srna generated from these three protein-coding, ppr genes were predicted to target to the mrnas of a number of p-class ppr genes  <cit> .
the gene expression signals of these three potential tasirna generating ppr genes  were indeed suppressed in the leaves of both oe lines in microarray studies .
there are  <dig> ppr genes in the arabidopsis genome  <cit> .
the transcript abundance of  <dig> and  <dig> ppr genes were significantly  changed in the leaves and the roots of both oe lines  .
the mir173-tasirnas-ppr/tpr network should play a role in the regulation of a specific group of ppr genes.
ppr
genes that had significant changes between both two oe lines and wild-type
pap2
fc: fold change.
atpap <dig> is targeted to both chloroplasts and mitochondria.
its overexpression resulted in a higher mir <dig> abundance  and tasirna biogenesis  from tas <dig> and tas <dig> loci , which may degrade their ppr/tpr target mrnas, as reflected by the microarray data .
ppr proteins, characterized by tandemly arranged 35-amino acid repeats, are predominantly targeted to chloroplasts or mitochondria and take part in virtually all the processes in rna processing and editing  <cit> .
further studies are required to understand how atpap <dig> overexpression affects ppr.
yeast-two-hybrid experiments showed that atpap <dig> can interact with a number of multiple organellar rna editing factors  , which interact with ppr proteins to carry out rna processing in chloroplasts and mitochondria  <cit> .
overexpression of atpap <dig> may modulate certain rna processing mechanisms in chloroplasts and mitochondria, thereby affecting the physiology of these two energy-generating organelles .
rna-seq of organeller rna from the overexpression line will give us a better picture on this hypothesis.
anthocyanin production can be induced in arabidopsis by stresses such as phosphate deficiency  <cit>  and sugar treatments  <cit> .
phosphate deficiency induces myb transcription factors , which subsequently activate the expression of multiple genes involved in anthocyanin synthesis.
myb <dig> also stimulates the expression of mir <dig>  which initiates the production of tas4-sir <dig> from the tas <dig> locus.
tas4-sir <dig> targets mrnas of myb transcription factors for cleavage and serves as a negative feedback loop of anthocyanin production  <cit> .
in this study, the counts of mir <dig> are zero in all the four lines, whereas the basal abundance of tas4-sir <dig> is lower in the oe lines  than the wt .
microarray studies showed that the basal abundance of many genes of enzymes involved in anthocyanin synthesis decreased in the leaves of the oe lines, while the transcript abundance of myb75/90/ <dig> was indifferent  <cit> .
these lower basal levels are physiologically relevant, as high sucrose  treatment induced less anthocyanin production in the oe lines than in the wt  <cit> .
phosphate deficiency induced all the three myb factors   <cit> , but only exogenous sucrose treatment induced myb <dig>  <cit> .
in the lines, high endogenous sugars suppress the basal transcript abundance of enzymes, which are independent of the transcript abundance of the myb transcription factors  <cit> .
these data indicates that the induction of anthocyanin production by exogenous sucrose and phosphate deficiency are overlapped but distinguishable.
in addition, since the count of mir <dig> is zero in all our samples, the cleavage of tas <dig> rna maybe initiated by an uncharacterized srna, adding complexity to the regulatory pathway of anthocyanin production.
CONCLUSIONS
energy is the driving force of growth; various biological processes are regulated by the availability of energy.
the atpap <dig> overexpression lines provide a unique opportunity to study the impact of high energy status on the regulatory mechanisms of plants.
our previous works showed that there are massive changes in the gene transcription profiles in the oe lines  <cit> .
small rna -mediated cleavage of mrna is the key mechanism to induce mrna degradation.
the present study is the first to report the srna expression profile of arabidopsis when ample supply of energy is available.
significant changes in certain mirnas and the mir173-tasirnas-ppr/tpr network were observed in the fast-growing lines.
further investigation is required to delineate the roles of the mir173-tasirnas network in plant growth and energy metabolism.

 small rna silencing is an essential mechanism in gene regulation in eukaryotes. there are two main categories of small rnas: micrornas and small interference rnas, which are generated from single-stranded, self-complementary rna transcripts and double-stranded rnas, respectively. the primary transcript of a mir gene is called pri-mirna, which is further processed into the stem-loop precursor mirna by dicer like. while the guide strands of the mirna duplexes are incorporated into argonaute of the rna-induced silencing complex, the passenger strands called mirna star are mostly degraded. the sources of dsrnas that trigger sirnas biogenesis could be exogenous or endogenous. plant evolved several classes of endogenous sirnas including tasirnas, natsirnas and cis-acting sirnas. in plants, tasirnas are generated by a pathway different from that of mirnas. the genomic loci encoding tasirnas are known as tas genes and are transcribed by polii. the generation of tasirna is initiated by mirna-mediated cleavage of long non-coding transcripts of tas genes. eight tas loci from four families are identified in arabidopsis genome. there are three loci in tas family, tas1a, tas1b and tas1c. both tas and tas transcripts are cleaved by mir and associated with ago to generate sirnas, which mainly target pentatricopeptide repeat-containing mrnas. there are three tas loci in arabidopsis thaliana, tas3a, tas3b and tas3c. mir guides cleavage of these transcripts with ago to generate sirnas which target mrnas of auxin responsive factors family. tas transcript is initiated by mir in association with ago to generate tasirnas and their targets are myb transcription factors. the cleaved rnas from the eight loci are bounded by suppressor of gene silencing and copied into dsrnas by rna-dependent rna polymerase. the dsrnas are cleaved in multiple rounds by dcl from the end defined by mirna-mediated cleavage such that the tasirnas are in 21-nucleotide register from the cleavage site. the tasirnas are loaded into ago complex to initiate tasirna guided mrna degradation. another class of sirnas is nat-sirnas, which could be derived from rnas transcribed from opposite strands of the same loci or by transcripts from different loci. there are and potential cis- and trans- natural antisense transcripts, respectively in arabidopsis. the production of nat-sirnas are is dependent on rdr and dcl or dcl. overexpression of atpap in a. thaliana speeds up plant growth. the oe lines flower early and grow faster than the wild type plants. the seed yield and silique numbers of oe lines are also more than the control lines. atpap was shown to be dually targeted to chloroplasts and mitochondria. metabolomics studies showed that some sugars, tca metabolites and certain amino acids are significantly higher in the oe lines than in the wt. the concentrations of atp and adp are also higher in the oe lines. all of these phenotypes pointed to a dramatic shift of metabolism in the overexpression lines. besides, overexpression of atpap in another member of brassicaceae can also generate fast growing and higher seed-yield transgenic plants. microarray data showed that thousands of transcripts were significantly altered in the oe lines, including key genes involved in carbon flow, k uptake and nitrogen assimilation. in addition, the transcription of many genes involved in mirnas and sirnas biogenesis was altered. the transcript abundance of key genes involved in mirna biogenesis, including dcl and hyl significantly increased in both oe lines, whereas sdn responsible for mirna degradation, had transcript abundance decreased in both oe lines. the transcript abundance of essential genes involved in tasirna biogenesis, including sgs ago and ago also significantly increased in both oe lines. to examine the small rnas profiles in the fast-growing lines, we constructed eight srna libraries from both leaves and roots of wild type, pap, and two independent overexpression lines. this study provides information on the impacts of high energy status on small rna profiles in arabidopsis. pap2, oe and oe21 pap2 *significantly changed in both oe lines. fc: fold change. the following genes were not significantly different between the wt and oe lines: ago ago ago dcl dcl hen hen hen hasty, nrpd1a, rdr rdr, sde and sde  small rnas length distribution and annotation eight small rna libraries were generated from the leaves and the roots of 20-day-old arabidopsis. after removal of adaptors and low quality tags, the number of cleans reads were calculated and mapped to the a. thaliana genome. in total, to million raw reads were generated from each library, and approximately 95% of the reads remained for further research after clearing the adaptors sequences. approximately to million sequence reads corresponding to to million unique sequence signatures could be mapped onto the genome. the length of the clean reads ranged from to nucleotides. the size distribution of small rna sequences in various leaf and root samples were reported in additional file similar distributions were observed in oe, pap and wild type leaf libraries. consistent with the earlier reports, two major peaks appeared at and nucleotides in length in the total sequence reads. in the root libraries, an additional peak at nucleotides was observed. these 19-nucletide sequences are mainly originated from trnas, and they are cleaved from the 5′ end of gly-trnatcc which represented over 80% of trna-derived small rnas in the roots. interestingly, the total reads of these 19-nucletide sequences are relatively more in both oe lines than in wt and pap. clean reads from the eight libraries were mapped and classified according to the annotation of the genome. a significant higher proportion of small rnas were mapped to trna genes in the root libraries than in the leaf libraries. for the repeat small rnas, both unique and total reads are less in oe leaf and root samples. we observed that the total reads of srna antisense to exons are higher in leaves of the oe lines, while there are smaller number of unique reads; these indicate that the srna antisense to exons are more concentrated to their targets in the leaves of oe lines. identification of known mirnas it has been reported that high throughput sequencing can be an alternative to estimate the expression profiles of mirna genes. this method allows us to distinguish the transcript abundance of the same mirna gene between different lines. small rnas were mapped to the mirna precursor/mature mirna species in mirbase. expression profiles of all known mirnas of the eight libraries were listed including mirnas. scatter plot showed the differential expression of mirna between different lines. the mirnas expression patterns between the two oe lines are indifferent, while comparing to wt as controls, there are significant changes. table  shows the mirnas with significant differential expression between the oe lines and the wt. all the known mirnas were calculated by fold change log > or < - and p-value <. the mirnas significantly changed in the oe lines mainly target to the mrnas of several protein families including spl family, hap2-like transcription factors, apetala 2-like transcription factors, tcp transcription factors, tas family, laccases, cation/hydrogen exchangers and jacalin lectins. these mirna expression data was cross-examined with the leaf microarray data. increases in some mirnas abundance correlates with the decreases in mrna transcript abundance, including ath-mir172a,b, ath-mir172c, ath-mir319a,b and ath-mir the targets are apetala2-like transcription factors, tcp transcription factors and jacalin lectins, respectively. the abundance of mir well known to be responsible for initiating tasirna biogenesis for tas1a, tas1b, tas1c and tas significantly increased in the leaves of both oe lines. in contrast, the abundance of mir a member of the mir family, significantly decreased in the leaves of both oe lines. pap2 pap2 numbers in bold mean significant changes in oe versus wt, p-value < and fold change log > or fold change log  < - all reads were normalized. the reads cut off was all the targets were predicted from website of mayer’s lab. analysis of novel mirnas candidates and their targets novel mirnas and their target genes were predicted by the mireap software. target genes are predicted based on the rules suggested by allen et al. and schwab. secondary structures were predicted and analyzed for stable stem-loop hairpins. the detail of novel mir genes including location, minus free energy, sequence and structures are listed in additional file according to the criteria described in methods, seven novel mirnas with counts >  were identified in leaf libraries, of which two were also identified in root libraries. for leaf/root_mirna0001rna_5p, it was predicted to target to chromomethylase. and for leaf_mirna0002_5p and root_mirna0002_3p, only found in wt/pap were predicted to target retrotransposon orf- protein. leaf_mirna0003_3p and leaf_mirna008_5p, which had relatively high reads, were only found in pap and wt, respectively and their targets were six f-box family proteins. leaf_mirna0005_5p and leaf_mirna0006_3p were predicted to target pentatricopeptide repeat family proteins, both are proteins located in mitochondria. these two mirnas were either not found or the counts were too low in the leaves of wt and pap line and therefore not detected in previous studies. trans-acting small interference srnas analysis the mapping of all the clean reads to the eight tasirnas loci, 21-nt tasirna reads were normalized and compared between samples. there were significant changes in the tasirna profiles in the leaves of both oe lines. as stated above, the counts of mir were significantly higher in the leaves of both oe lines. mir was known to induce cleavage of tas and tas transcripts and initiate tasirnas production from these loci. our data shows that the amounts of tasirnas generated from tas1a, b, c and tas were significantly increased in the leaves of the oe lines. phase register and count distribution of tas1a, tas1b, tas1c and tas sites cleaved by mir in leaf were presented. the phase distribution pattern of tas1b was significantly altered between oe and wt lines. the predicted targets of many of these tasirna are mrnas of ppr and tpr genes. in the four leaf samples, individual 21-nt srna sequences with reads > could be mapped to the four tas and tas loci, of which srna sequences were found to be significantly differentially expressed between the wt and both oe lines. the same srna sequences could be generated from more than one locus, and multiple srnas are predicted to target to the same target. a network of mir173-tasirnas-ppr/tpr was generated to show their relationship. all the potential ppr/tpr genes that appeared in the network were listed in additional file for tas the abundance of tasirna tas4-sir strongly decreased in both oe lines compared to the wt. in contrast, there are no significant changes in the tasirnas generated from the three tas loci. natural-antisense-transcript derived small interference rnas out of potential cis-nats predicted from arabidopsis tair database, we could only map our srnas to cis-nats; only and pairs were significantly altered in the leaves and roots of both oe lines. only cis-nats increased in abundance in the leaves of the oe lines, some have significant fold changes. the genes in the gene pairs include mirnas, tas1c, one ppr gene, two genes involved in auxin response, a histone h2a gene and a phosphoribosylanthranilate isomerase gene. the srna counts for the latter two gene pairs increased by 10- and 60-fold in leaves, respectively. some cis-nats strongly decreased in abundance in the leaves of oe lines. the genes in the gene pairs include genes of lhcb, lhcb a protein kinase apk2b, 2 f-box proteins and a udp-3-o- n-acetylglucosamine deacetylase. the srna mapped to the latter gene pair dropped ~100-fold in both leaves and roots. out of possible trans-nats pairs, only and pairs were significantly altered in the leaves and roots of both oe lines, respectively. most of the genes involved are transponsable elements, pseudogenes and genes for unknown proteins. validation of sirnas and candidate genes by qrt-pcr to validate the srna sequencing data, the abundance of selected mirnas and 21-nt tasi-rnas mapped to cds of tasib, tas1c, tas and tas4-sir in different lines were compared by qrt-pcr. all the results of selected small rnas were consistent between qrt-pcr and sequencing. the microarray data has been validated by qrt-pcr in our previous study and the transcript abundance of selected genes, including sgs at1g, were also well validated by qrt-pcr in this study. discussion srna biogenesis is one of the regulatory mechanisms in organism. in this study, the srna profiles of high energy plants were compared with that of wt plants under soil-grown condition. the size and growth stages of the plants were identical so that variations due to developmental stages and morphology were minimized. many mirnas and sirnas were activated by various biotic and abiotic stresses and subsequently modulated the mrna stability of their target genes, so that the plants can cope with the stresses accordingly. our data shows that very few mirnas were significantly altered in the oe lines under the experimental conditions. out of known mirnas in arabidopsis, only and mirnas significantly increased or decreased in abundance in the leaves of oe lines. very few mirnas were altered in the roots, only and mirnas significantly increased or decreased in abundance, respectively. some mirnas were shown to control plant development. overexpression of mir and mir resulted in late-flowering, and overexpression of mir resulted in increased lateral rooting. however, the abundance of these mirnas was unaltered in the oe lines. only the abundance of mir and mir were significantly higher in the leaves of the oe lines. the targets of mir and mir are ap and tcp, transcription factors, respectively. both are involved in leaf development, floral organ identity and flowering time. overexpression of mir in arabidopsis and potato can promote flowering development and tuberization respectively. the higher expression of mir in the leaves of oe lines correlates with the earlier bolting and flowering phenotypes of the oe lines. for mirnas that are induced by nutritional stresses, such as phosphate deficiency copper deficiency, and sulfate deficiency, their abundance were low in all lines and were not significantly changed in our oe lines. this is reasonable as the plants were grown in soil with adequate supply of nutrients. similarly, mirnas responsive to bacterial and viral infections were not altered in the oe lines. the abundance of some mirna induced by cold stresses increased in the oe lines but many other mirnas induced by cold were unaltered. accumulation of sucrose in the cytosol is a key protection mechanism for cold tolerance. since the oe lines contain higher sucrose contents, mir and mir might be induced indirectly by cold-induced sucrose accumulation. currently, eight tas genes of tasirnas have been identified in the arabidopsis genome. the transcribed mrnas from these loci require mirna-induced cleavage to generate functional tasirnas, which in turn induce the cleavage of the target mrnas. for example, the tas1a/b/c and tas tasirna families are induced by mir which then down-regulate mrnas of various ppr and tpr genes.our data shows that the mir173-tasirnas-ppr/tpr network had substantial changes in the leaves of both oe lines. a tas gene prediction algorithm predicted seven tas loci in a. thaliana using col- mpss small rna data, which include tas1a/b/c, tas and ppr genes. srna generated from these three protein-coding, ppr genes were predicted to target to the mrnas of a number of p-class ppr genes. the gene expression signals of these three potential tasirna generating ppr genes were indeed suppressed in the leaves of both oe lines in microarray studies. there are ppr genes in the arabidopsis genome. the transcript abundance of and ppr genes were significantly changed in the leaves and the roots of both oe lines. the mir173-tasirnas-ppr/tpr network should play a role in the regulation of a specific group of ppr genes. ppr genes that had significant changes between both two oe lines and wild-type pap2 fc: fold change. atpap is targeted to both chloroplasts and mitochondria. its overexpression resulted in a higher mir abundance and tasirna biogenesis from tas and tas loci, which may degrade their ppr/tpr target mrnas, as reflected by the microarray data. ppr proteins, characterized by tandemly arranged 35-amino acid repeats, are predominantly targeted to chloroplasts or mitochondria and take part in virtually all the processes in rna processing and editing. further studies are required to understand how atpap overexpression affects ppr. yeast-two-hybrid experiments showed that atpap can interact with a number of multiple organellar rna editing factors, which interact with ppr proteins to carry out rna processing in chloroplasts and mitochondria. overexpression of atpap may modulate certain rna processing mechanisms in chloroplasts and mitochondria, thereby affecting the physiology of these two energy-generating organelles. rna-seq of organeller rna from the overexpression line will give us a better picture on this hypothesis. anthocyanin production can be induced in arabidopsis by stresses such as phosphate deficiency and sugar treatments. phosphate deficiency induces myb transcription factors, which subsequently activate the expression of multiple genes involved in anthocyanin synthesis. myb also stimulates the expression of mir which initiates the production of tas4-sir from the tas locus. tas4-sir targets mrnas of myb transcription factors for cleavage and serves as a negative feedback loop of anthocyanin production. in this study, the counts of mir are zero in all the four lines, whereas the basal abundance of tas4-sir is lower in the oe lines than the wt. microarray studies showed that the basal abundance of many genes of enzymes involved in anthocyanin synthesis decreased in the leaves of the oe lines, while the transcript abundance of myb75/90/ was indifferent. these lower basal levels are physiologically relevant, as high sucrose treatment induced less anthocyanin production in the oe lines than in the wt. phosphate deficiency induced all the three myb factors, but only exogenous sucrose treatment induced myb. in the lines, high endogenous sugars suppress the basal transcript abundance of enzymes, which are independent of the transcript abundance of the myb transcription factors. these data indicates that the induction of anthocyanin production by exogenous sucrose and phosphate deficiency are overlapped but distinguishable. in addition, since the count of mir is zero in all our samples, the cleavage of tas rna maybe initiated by an uncharacterized srna, adding complexity to the regulatory pathway of anthocyanin production.  energy is the driving force of growth; various biological processes are regulated by the availability of energy. the atpap overexpression lines provide a unique opportunity to study the impact of high energy status on the regulatory mechanisms of plants. our previous works showed that there are massive changes in the gene transcription profiles in the oe lines. small rna -mediated cleavage of mrna is the key mechanism to induce mrna degradation. the present study is the first to report the srna expression profile of arabidopsis when ample supply of energy is available. significant changes in certain mirnas and the mir173-tasirnas-ppr/tpr network were observed in the fast-growing lines. further investigation is required to delineate the roles of the mir173-tasirnas network in plant growth and energy metabolism. 
mirnas with increased abundance in the leaf and root samples of both oe lines were predicted to target mrnas coding for dof zinc finger protein and apetala proteins, respectively. in the leaf of oe lines, known mirnas increased in abundance and mirnas decreased in abundance, whereas in the root of oe lines, known mirnas increased in abundance and mirnas decreased in abundance. furthermore, a significant change in the mir173-tasirnas-ppr/tpr network was observed in the leaves of both oe lines. an induction of mir173-tasirnas-ppr/tpr network was also observed in the oe lines. overexpression lines of arabidopsis thaliana purple acid phosphatase were shown to grow faster and exhibit higher atp and sugar contents. in this study, the srna profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on srna expression were analyzed. in this study, the impact of high energy content on the srna profiles of arabidopsis is reported. in higher eukaryotes, small rnas play a role in regulating gene expression.
 in higher eukaryotes, small rnas play a role in regulating gene expression. overexpression lines of arabidopsis thaliana purple acid phosphatase were shown to grow faster and exhibit higher atp and sugar contents. leaf microarray studies showed that many genes involved in micrornas and trans-acting sirnas biogenesis were significantly changed in the fast-growing lines. in this study, the srna profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on srna expression were analyzed.  9- million reads from each library were mapped to genome. mirnas, tasirnas and natural antisense transcripts-generated small interfering rnas were identified and compared between libraries. in the leaf of oe lines, known mirnas increased in abundance and mirnas decreased in abundance, whereas in the root of oe lines, known mirnas increased in abundance and mirnas decreased in abundance. mirnas with increased abundance in the leaf and root samples of both oe lines were predicted to target mrnas coding for dof zinc finger protein and apetala proteins, respectively. furthermore, a significant change in the mir173-tasirnas-ppr/tpr network was observed in the leaves of both oe lines.  in this study, the impact of high energy content on the srna profiles of arabidopsis is reported. while the abundance of many stress-induced mirnas is unaltered, the abundance of some mirnas related to plant growth and development is elevated in the fast-growing lines. an induction of mir173-tasirnas-ppr/tpr network was also observed in the oe lines. in contrast, only few cis- and trans-natsirnas are altered in the fast-growing lines. chloroplastsmitochondriamirnasmorfpprtasirnas and natsirnas 