BACKGROUND
as one of the important organs, the root system helps a plant to anchorage to the soil, and to absorb mineral nutrients and water for normal development and growth during its life cycle.
since the root system architecture greatly affects the efficiency of nutrient and water absorption, the molecular factors influencing root development have been extensively investigated in order to facilitate genetic modification of the agronomic traits of the plants, especially for the crops  <cit> .
micrornas , a small rna  species, were identified to be involved in various biological processes of plants during the past ten years  <cit> .
certain mirna families are indispensable for the establishment of the root system architecture  <cit> , and are implicated in the transduction of multiple signals, such as nutrition, hormone and stress  <cit> .
for examples, mir <dig> is required for root cap formation by targeting auxin response factor   <dig> and  <dig> in arabidopsis <cit> .
mir164-mediated regulation of nam/ataf/cuc  <dig>  is involved in arabidopsis lateral root development through downregulating auxin signals  <cit> .
both mir <dig> and mir <dig> play important roles in adventitious rooting in arabidopsis <cit> .
based on high-throughput sequencing , a much more huge srna population was uncovered in plants in addition to the mirnas.
whether some of these srnas play potential roles in root development remains unclear, although a few experimental hints point to this possibility recently  <cit> .
to partially address this issue, we took advantage of the public srna hts data originated from the root tips and the whole roots of rice .
we made a comparison of the srna accumulation levels between the two biological samples to identify the srnas highly accumulated in the root tips and in the whole roots respectively.
then, these srnas were subjected to argonaute  <dig>  enrichment analysis.
and, those enriched in ago <dig> were considered to have potential of performing target cleavages, and were included in target prediction and degradome sequencing data-based validation.
based on the target lists, regulatory networks mediated by the ago1-enriched srnas were established for both the root tips and the whole roots.
notably, within the networks, the expression patterns of several target genes, based on the microarray data, were contrary to those of the regulating srnas, supporting the interactions between the srnas and the targets.
besides, literature mining indicated that certain target genes played potential roles in root development.
these evidences indicate the reliability of the networks.
our results could advance the current understanding of the regulatory roles of the srnas, in addition to the mirnas, in plant root growth and development.
RESULTS
identification of the srnas highly accumulated in the root tips or in the whole roots of rice
we took advantage of the public srna hts data of rice.
the data sets could be divided into two groups: the ones  prepared from the rice root tips  and the ones  from the whole roots .
according to the previous reports  <cit> , all of these data sets originated from ~14-day-old rice  seedlings, thus making the two data groups more comparable.
to extract the srnas highly accumulated in the root tips, the following rules were applied:  the srna should be present in at least one of the “root tip” data sets ;  its normalized accumulation levels should be  <dig> rpm  or higher, and should be three times or more than the levels of the same sequences in all the “whole root” data sets .
similar rules were applied to identify the srnas highly accumulated in the whole roots.
as a result,  <dig>  srnas including eight mirna sequences  showed high abundances in the root tips , and  <dig>  srnas including  <dig> mirna sequences  were highly accumulated in the whole roots .
since the “whole root” includes the “root tip” cells, we reasoned that the high levels of the  <dig>  srnas in the whole roots were largely attributed to their high abundances within the elongation and the maturation zones of the rice roots including the main roots, the adventitious roots and the lateral roots.
in plants, some of the srnas, such as the mirnas, exert their regulatory activities through target cleavages  <cit> .
the srnas incorporate into specific ago-associated rna-induced silencing complexes , and guide the complexes to the target transcripts to perform cleavages.
it has been reported that ago <dig> is an rna slicer selectively recruiting mirnas and short interfering rnas in plants  <cit> .
in this consideration, we set out to identify ago1-enriched srnas from the srnas highly accumulated in the root tips and the whole roots obtained above.
the following rules were applied:  the srnas should be detectable in at least one of the ago1-associated srna hts data sets ;  its normalized accumulation levels should be  <dig> rpm or higher, and should be three times or more than the levels of the same sequence in the control set .
as a result,  <dig> ago1-enriched srnas were extracted from the srnas highly accumulated in the root tips, and  <dig> ago1-enriched srnas including osa-mir528-5p were identified from the srnas highly accumulated in the whole roots .
next, the sequence characteristics of the srnas before and after the ago <dig> enrichment filtering were examined separately.
for both the srnas highly accumulated in the root tips and those highly abundant in the whole roots, no significant bias of the 5′ terminal nucleotide compositions was observed before the ago <dig> enrichment filtering .
however, for those enriched in ago <dig> complexes, the proportion of 5′ u -started srnas was increased.
specifically, for the srnas highly accumulated in the whole roots, the percentage of 5′ u-started srnas increased from  <dig> % to  <dig> %.
for those highly accumulated in the root tips, the percentage increased from  <dig> % to  <dig> % .
different from the mirnas,  <dig> nt  is not the dominant length for the other srnas highly accumulated in the whole roots.
more interestingly,  <dig> nt is the dominant length for both the srnas and the mirnas highly accumulated in the root tips .
after the ago <dig> enrichment filtering, the srnas ranging from  <dig> nt to  <dig> nt occupied a dominant portion  of the srnas abundant in the whole roots.
and, for the srnas highly accumulated in the root tips, the  <dig> nt ones increased significantly  .
considering the previous report on the sequence characteristics of ago1-associated srnas  <cit> , our ago <dig> enrichment filtering tended to selectively extract 5′ u-started,  <dig> to  <dig> nt srnas from the srnas highly accumulated in the root tips and the whole roots, respectively.
identification of the targets of the ago1-enriched srnas and network construction
as mentioned above, the srnas associated with the ago <dig> silencing complexes are more likely to modulate gene expression through target cleavages.
in this analysis, only the ago1-enriched srnas were subjected to target prediction.
miru algorithm  <cit>  with default parameters was employed for this prediction.
as a result,  <dig>  target binding sites were predicted for the  <dig> ago1-enriched srnas highly expressed in the root tips, and  <dig>  binding sites were predicted for the  <dig> ago1-enriched srnas  highly expressed in the whole roots.
degradome sequencing data is a valuable resource for large-scale validation of the srna—target interactions  <cit> .
four degradome sequencing data sets of rice  were recruited to do a comprehensive validation of the predicted targets according to the analytical workflow proposed previously  <cit> .
considering the reports that some evident cleavage signals out of the canonical regions  were observed  <cit> , the binding sites with prominent cleavage signals resided within 8th to 12th nt of the regulating srnas were retained.
after target plot-based screening,  <dig> interactions involving  <dig> target genes and six srnas highly accumulated in the root tips, and  <dig> interactions involving  <dig> genes and  <dig> srnas  highly abundant in the whole roots passed the validation .
among the validated srna—target pairs, we observed that many binding sites were supported by evident degradome signatures resided within the canonical cleavage sites  .
in addition to osa-mir528-5p , several srnas, such as rt_high_srna <dig>  rt_high_srna <dig>  rt_high_rna <dig>  wr_high_srna <dig> and wr_high_srna <dig>  cleaved the corresponding transcripts at the canonical sites.
for wr_high_srna <dig>  we observed that the srna possessed two binding sites on the transcripts loc_os01g <dig> , loc_os01g <dig>  and loc_os05g <dig> , separately .
notably, all of these srnas are distinct from the current plant mirna registries in mirbase   <cit> .
the result indicates that the srnas, which remain to be classified, could guide ago <dig> complexes to perform mirna-like target cleavages in the root systems of rice.
based on the target lists obtained above, the srna-mediated regulatory networks were constructed in the root tips and the whole roots by using cytoscape  <cit>  .
at first glance, rt_high_srna <dig> regulates  <dig> transcripts encoded by ten different genes , pointing to the possibility that this srna might play an essential role in root tip formation.
more complex network was observed for those srnas highly accumulated in the whole roots.
in some cases, one target is shared by two or more srnas.
for example, loc_os07g <dig>  is regulated by wr_high_srna <dig> and wr_high_srna <dig>  loc_os09g <dig>  is regulated by wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig> and wr_high_srna <dig> .
biological meanings of the networks supported by target expression- and literature mining-based evidences
considering the repressive role of the ago1-enriched srnas in gene expression, we deduced that for the srna—target pairs validated above, the target genes should display expression patterns contrary to those of the srna regulators.
for example, the target gene of a srna highly accumulated in the root tips was supposed to have higher expression level in the whole roots than in the root tips.
to support this notion,  <dig> microarray data sets contributed by a previous report  <cit>  were utilized.
these data sets enabled us to investigate the expression levels of all the target genes in different parts of the roots .
according to the report by takehisa et al.
<cit> , in this analysis, the microarray data sets of the root cap and the division zone were grouped into the “root tip” category , which was anatomically comparable to the srna hts library group  prepared from the root tips .
all the  <dig> microarray data sets were treated as the “whole root” category in order to make it anatomically consistent with the srna hts library group  prepared from the whole roots.
all the microarray data sets were generated from the 10-day-old rice seedlings, which was largely consistent with the 14-day-old seedlings for srna library preparation.
for the expression level analysis, the mean value of the three replicates was calculated for each target gene.
the microarray data-based analysis provided supportive evidences for some of the srna—target interactions.
specifically, the level of loc_os02g <dig>  regulated by rt_high_srna <dig> in the whole roots is  <dig>  times higher than its level in the root tips  .
for loc_os07g <dig> /loc_os07g <dig> /loc_os07g <dig>  cleaved by rt_high_srna <dig>  their expression value in the whole roots is  <dig>  versus  <dig>  in the root tips.
also regulated by rt_high_srna <dig>  the level of loc_os09g <dig>  in the whole roots is  <dig>  versus  <dig>  in the root tips .
for loc_os04g <dig> /loc_os04g <dig>  regulated by wr_high_srna <dig>  their expression value in the whole roots is  <dig> , which is lower than that  in the root tips.
for loc_os06g <dig> /loc_os06g <dig>  also regulated by wr_high_srna <dig>  their expression value in the whole roots is  <dig>  versus  <dig>  in the root tips.
also for wr_high_srna <dig>  the level of its target transcript loc_os12g <dig>  in the whole roots is  <dig> , which is lower than that  in the root tips .
for loc_os09g <dig>  regulated by wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig> and wr_high_srna <dig>  its expression levels in the whole roots and the root tips are  <dig>  and  <dig>  respectively .
taken together, the results demonstrated that at least some of the target genes displayed expression patterns contrary to those of the regulating srnas.
failure to validate the regulatory relationships of the remaining srna—target pairs by microarray data analysis might attribute to several embedded factors.
the most likely reason might be that the expression of the target genes was not only modulated by the identified regulatory srnas, but also regulated by the other factors such as transcription factors and epigenetic factors which might disturb the contrary accumulation patterns between the srnas and the targets in the root systems.
from this point of view, we should recognize that the regulation of the expression of a gene is quite complex, and could not be determined by only one regulator in most cases.
another potential reason is that the target gene might be specifically expressed in the root part in which the regulating srnas was highly abundant.
for example, the srna was highly abundant in the root tips, and the target gene was expressed in the root tips but not in the remaining parts of the rice roots.
it will lead to the failure to observe the contrary accumulation pattern between this srna—target pair.
we also intended to find some root-related evidences for the target genes based on functional annotations and literature mining.
fortunately, some positive hints were obtained.
for examples, based on the annotation from tigr rice   <cit> , loc_os02g <dig>  regulated by rt_high_srna <dig>  is involved in post-embryonic development.
and, its homologous gene in arabidopsis, at5g <dig>  participates in the organization of microtubules during cell division according to the tair  annotation  <cit> .
since the root tip includes a meristematic region in an active state of cell division, whether loc_os02g <dig> plays an essential role in the formation and/or the maintenance of the root meristem remains to be investigated.
also in the root tips, loc_os11g <dig> encoding an f-box domain-containing protein is regulated by rt_high_srna <dig> based on the degradome sequencing data .
according to a recent report, an f-box gene loc_os02g <dig> is involved in promoting root growth in rice  <cit> , pointing to the possibility that loc_os11g <dig> is functionally linked to root development.
more supportive evidences were found for the target genes within the network mediated by the srnas abundant in the whole roots.
loc_os03g <dig>  loc_os03g <dig>  loc_os10g <dig> and loc_os12g <dig>  all of which encode start domain-containing proteins , were regulated by the same srna regulators, i.e.
wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig>  wr_high_srna <dig> and wr_high_srna <dig> .
in arabidopsis and medicago truncatula, mir166-mediated regulation of class iii hd-zip genes is important for root development  <cit> .
more interestingly, although the above nine srnas are not mirnas, wr_high_srna <dig> shares high sequence similarity with osa-mir166g-3p and osa-mir166h-3p of rice, and the other eight srnas share high sequence similarity with mir <dig> of the other plant species.
in this regard, the four target genes regulated by the srnas homologous to mir <dig> might have a conserved role in rice root development.
another class of target genes including loc_os04g <dig>  loc_os06g <dig> and loc_os12g <dig> encode the rice arf proteins  .
the three arf genes share high sequence identity with each other, and loc_os04g <dig> was reported to regulate root elongation in rice  <cit> .
moreover, the homologous arf genes in arabidopsis are regulated by mir <dig>  and are essential for root system development  <cit> .
notably, the three arf genes of rice were targeted by wr_high_srna <dig> sharing high sequence similarity with osa-mir <dig>  loc_os06g <dig> targeted by osa-mir528-5p encodes osfbl <dig>  an f-box domain- and lrr-containing protein .
its homologous gene at2g <dig>  is implicated in root development in arabidopsis <cit> .
and, mir <dig> was previously indicated to play a role in auxin signal-mediated rice root development  <cit> .
loc_os09g <dig> encoding an hmg-y-related protein was targeted by five srnas highly accumulated in the whole roots .
based on an early report, ectopic expression of the maize hmgb <dig> protein causes defects in root development of tobacco seedlings  <cit> .
more direct evidence was found for loc_os07g <dig> targeted by wr_high_srna <dig>  sharing sequence similarity with mir <dig> of the other plant species.
this target gene encodes glr   <dig>  based on the tigr annotation.
it was reported that glr <dig>  of rice was essential for the maintenance of the root apical meristem  <cit> , and glr <dig>  and glr <dig>  of arabidopsis were involved in lateral root initiation  <cit> .
all these literature-based evidences support the potential roles of the target genes in rice root development.
CONCLUSIONS
summarily, this study on the srnas in the rice root systems could advance our knowledge of the regulatory activities and the biological functions of the plant srnas other than the mirnas.
the networks established in this study were supported by the degradome sequencing data, the microarray data and the literature-based evidences, indicating that the networks could provide a basis for in-depth analysis of srna-mediated gene regulation in the rice roots.

 as one of the important organs, the root system helps a plant to anchorage to the soil, and to absorb mineral nutrients and water for normal development and growth during its life cycle. since the root system architecture greatly affects the efficiency of nutrient and water absorption, the molecular factors influencing root development have been extensively investigated in order to facilitate genetic modification of the agronomic traits of the plants, especially for the crops. micrornas, a small rna species, were identified to be involved in various biological processes of plants during the past ten years. certain mirna families are indispensable for the establishment of the root system architecture, and are implicated in the transduction of multiple signals, such as nutrition, hormone and stress. for examples, mir is required for root cap formation by targeting auxin response factor and in arabidopsis. mir164-mediated regulation of nam/ataf/cuc is involved in arabidopsis lateral root development through downregulating auxin signals. both mir and mir play important roles in adventitious rooting in arabidopsis. based on high-throughput sequencing, a much more huge srna population was uncovered in plants in addition to the mirnas. whether some of these srnas play potential roles in root development remains unclear, although a few experimental hints point to this possibility recently. to partially address this issue, we took advantage of the public srna hts data originated from the root tips and the whole roots of rice. we made a comparison of the srna accumulation levels between the two biological samples to identify the srnas highly accumulated in the root tips and in the whole roots respectively. then, these srnas were subjected to argonaute enrichment analysis. and, those enriched in ago were considered to have potential of performing target cleavages, and were included in target prediction and degradome sequencing data-based validation. based on the target lists, regulatory networks mediated by the ago1-enriched srnas were established for both the root tips and the whole roots. notably, within the networks, the expression patterns of several target genes, based on the microarray data, were contrary to those of the regulating srnas, supporting the interactions between the srnas and the targets. besides, literature mining indicated that certain target genes played potential roles in root development. these evidences indicate the reliability of the networks. our results could advance the current understanding of the regulatory roles of the srnas, in addition to the mirnas, in plant root growth and development.  identification of the srnas highly accumulated in the root tips or in the whole roots of rice we took advantage of the public srna hts data of rice. the data sets could be divided into two groups: the ones prepared from the rice root tips and the ones from the whole roots. according to the previous reports, all of these data sets originated from ~14-day-old rice seedlings, thus making the two data groups more comparable. to extract the srnas highly accumulated in the root tips, the following rules were applied: the srna should be present in at least one of the “root tip” data sets; its normalized accumulation levels should be rpm or higher, and should be three times or more than the levels of the same sequences in all the “whole root” data sets. similar rules were applied to identify the srnas highly accumulated in the whole roots. as a result, srnas including eight mirna sequences showed high abundances in the root tips, and srnas including mirna sequences were highly accumulated in the whole roots. since the “whole root” includes the “root tip” cells, we reasoned that the high levels of the srnas in the whole roots were largely attributed to their high abundances within the elongation and the maturation zones of the rice roots including the main roots, the adventitious roots and the lateral roots. in plants, some of the srnas, such as the mirnas, exert their regulatory activities through target cleavages. the srnas incorporate into specific ago-associated rna-induced silencing complexes, and guide the complexes to the target transcripts to perform cleavages. it has been reported that ago is an rna slicer selectively recruiting mirnas and short interfering rnas in plants. in this consideration, we set out to identify ago1-enriched srnas from the srnas highly accumulated in the root tips and the whole roots obtained above. the following rules were applied: the srnas should be detectable in at least one of the ago1-associated srna hts data sets; its normalized accumulation levels should be rpm or higher, and should be three times or more than the levels of the same sequence in the control set. as a result, ago1-enriched srnas were extracted from the srnas highly accumulated in the root tips, and ago1-enriched srnas including osa-mir528-5p were identified from the srnas highly accumulated in the whole roots. next, the sequence characteristics of the srnas before and after the ago enrichment filtering were examined separately. for both the srnas highly accumulated in the root tips and those highly abundant in the whole roots, no significant bias of the 5′ terminal nucleotide compositions was observed before the ago enrichment filtering. however, for those enriched in ago complexes, the proportion of 5′ u -started srnas was increased. specifically, for the srnas highly accumulated in the whole roots, the percentage of 5′ u-started srnas increased from % to %. for those highly accumulated in the root tips, the percentage increased from % to %. different from the mirnas, nt is not the dominant length for the other srnas highly accumulated in the whole roots. more interestingly, nt is the dominant length for both the srnas and the mirnas highly accumulated in the root tips. after the ago enrichment filtering, the srnas ranging from nt to nt occupied a dominant portion of the srnas abundant in the whole roots. and, for the srnas highly accumulated in the root tips, the nt ones increased significantly. considering the previous report on the sequence characteristics of ago1-associated srnas, our ago enrichment filtering tended to selectively extract 5′ u-started, to nt srnas from the srnas highly accumulated in the root tips and the whole roots, respectively. identification of the targets of the ago1-enriched srnas and network construction as mentioned above, the srnas associated with the ago silencing complexes are more likely to modulate gene expression through target cleavages. in this analysis, only the ago1-enriched srnas were subjected to target prediction. miru algorithm with default parameters was employed for this prediction. as a result, target binding sites were predicted for the ago1-enriched srnas highly expressed in the root tips, and binding sites were predicted for the ago1-enriched srnas highly expressed in the whole roots. degradome sequencing data is a valuable resource for large-scale validation of the srna—target interactions. four degradome sequencing data sets of rice were recruited to do a comprehensive validation of the predicted targets according to the analytical workflow proposed previously. considering the reports that some evident cleavage signals out of the canonical regions were observed, the binding sites with prominent cleavage signals resided within 8th to 12th nt of the regulating srnas were retained. after target plot-based screening, interactions involving target genes and six srnas highly accumulated in the root tips, and interactions involving genes and srnas highly abundant in the whole roots passed the validation. among the validated srna—target pairs, we observed that many binding sites were supported by evident degradome signatures resided within the canonical cleavage sites. in addition to osa-mir528-5p, several srnas, such as rt_high_srna rt_high_srna rt_high_rna wr_high_srna and wr_high_srna cleaved the corresponding transcripts at the canonical sites. for wr_high_srna we observed that the srna possessed two binding sites on the transcripts loc_os01g, loc_os01g and loc_os05g, separately. notably, all of these srnas are distinct from the current plant mirna registries in mirbase. the result indicates that the srnas, which remain to be classified, could guide ago complexes to perform mirna-like target cleavages in the root systems of rice. based on the target lists obtained above, the srna-mediated regulatory networks were constructed in the root tips and the whole roots by using cytoscape. at first glance, rt_high_srna regulates transcripts encoded by ten different genes, pointing to the possibility that this srna might play an essential role in root tip formation. more complex network was observed for those srnas highly accumulated in the whole roots. in some cases, one target is shared by two or more srnas. for example, loc_os07g is regulated by wr_high_srna and wr_high_srna loc_os09g is regulated by wr_high_srna wr_high_srna wr_high_srna wr_high_srna and wr_high_srna. biological meanings of the networks supported by target expression- and literature mining-based evidences considering the repressive role of the ago1-enriched srnas in gene expression, we deduced that for the srna—target pairs validated above, the target genes should display expression patterns contrary to those of the srna regulators. for example, the target gene of a srna highly accumulated in the root tips was supposed to have higher expression level in the whole roots than in the root tips. to support this notion, microarray data sets contributed by a previous report were utilized. these data sets enabled us to investigate the expression levels of all the target genes in different parts of the roots. according to the report by takehisa et al., in this analysis, the microarray data sets of the root cap and the division zone were grouped into the “root tip” category, which was anatomically comparable to the srna hts library group prepared from the root tips. all the microarray data sets were treated as the “whole root” category in order to make it anatomically consistent with the srna hts library group prepared from the whole roots. all the microarray data sets were generated from the 10-day-old rice seedlings, which was largely consistent with the 14-day-old seedlings for srna library preparation. for the expression level analysis, the mean value of the three replicates was calculated for each target gene. the microarray data-based analysis provided supportive evidences for some of the srna—target interactions. specifically, the level of loc_os02g regulated by rt_high_srna in the whole roots is times higher than its level in the root tips. for loc_os07g /loc_os07g /loc_os07g cleaved by rt_high_srna their expression value in the whole roots is versus in the root tips. also regulated by rt_high_srna the level of loc_os09g in the whole roots is versus in the root tips. for loc_os04g /loc_os04g regulated by wr_high_srna their expression value in the whole roots is, which is lower than that in the root tips. for loc_os06g /loc_os06g also regulated by wr_high_srna their expression value in the whole roots is versus in the root tips. also for wr_high_srna the level of its target transcript loc_os12g in the whole roots is, which is lower than that in the root tips. for loc_os09g regulated by wr_high_srna wr_high_srna wr_high_srna wr_high_srna and wr_high_srna its expression levels in the whole roots and the root tips are and respectively. taken together, the results demonstrated that at least some of the target genes displayed expression patterns contrary to those of the regulating srnas. failure to validate the regulatory relationships of the remaining srna—target pairs by microarray data analysis might attribute to several embedded factors. the most likely reason might be that the expression of the target genes was not only modulated by the identified regulatory srnas, but also regulated by the other factors such as transcription factors and epigenetic factors which might disturb the contrary accumulation patterns between the srnas and the targets in the root systems. from this point of view, we should recognize that the regulation of the expression of a gene is quite complex, and could not be determined by only one regulator in most cases. another potential reason is that the target gene might be specifically expressed in the root part in which the regulating srnas was highly abundant. for example, the srna was highly abundant in the root tips, and the target gene was expressed in the root tips but not in the remaining parts of the rice roots. it will lead to the failure to observe the contrary accumulation pattern between this srna—target pair. we also intended to find some root-related evidences for the target genes based on functional annotations and literature mining. fortunately, some positive hints were obtained. for examples, based on the annotation from tigr rice, loc_os02g regulated by rt_high_srna is involved in post-embryonic development. and, its homologous gene in arabidopsis, at5g participates in the organization of microtubules during cell division according to the tair annotation. since the root tip includes a meristematic region in an active state of cell division, whether loc_os02g plays an essential role in the formation and/or the maintenance of the root meristem remains to be investigated. also in the root tips, loc_os11g encoding an f-box domain-containing protein is regulated by rt_high_srna based on the degradome sequencing data. according to a recent report, an f-box gene loc_os02g is involved in promoting root growth in rice, pointing to the possibility that loc_os11g is functionally linked to root development. more supportive evidences were found for the target genes within the network mediated by the srnas abundant in the whole roots. loc_os03g loc_os03g loc_os10g and loc_os12g all of which encode start domain-containing proteins, were regulated by the same srna regulators, i.e. wr_high_srna wr_high_srna wr_high_srna wr_high_srna wr_high_srna wr_high_srna wr_high_srna wr_high_srna and wr_high_srna. in arabidopsis and medicago truncatula, mir166-mediated regulation of class iii hd-zip genes is important for root development. more interestingly, although the above nine srnas are not mirnas, wr_high_srna shares high sequence similarity with osa-mir166g-3p and osa-mir166h-3p of rice, and the other eight srnas share high sequence similarity with mir of the other plant species. in this regard, the four target genes regulated by the srnas homologous to mir might have a conserved role in rice root development. another class of target genes including loc_os04g loc_os06g and loc_os12g encode the rice arf proteins. the three arf genes share high sequence identity with each other, and loc_os04g was reported to regulate root elongation in rice. moreover, the homologous arf genes in arabidopsis are regulated by mir and are essential for root system development. notably, the three arf genes of rice were targeted by wr_high_srna sharing high sequence similarity with osa-mir loc_os06g targeted by osa-mir528-5p encodes osfbl an f-box domain- and lrr-containing protein. its homologous gene at2g is implicated in root development in arabidopsis. and, mir was previously indicated to play a role in auxin signal-mediated rice root development. loc_os09g encoding an hmg-y-related protein was targeted by five srnas highly accumulated in the whole roots. based on an early report, ectopic expression of the maize hmgb protein causes defects in root development of tobacco seedlings. more direct evidence was found for loc_os07g targeted by wr_high_srna sharing sequence similarity with mir of the other plant species. this target gene encodes glr based on the tigr annotation. it was reported that glr of rice was essential for the maintenance of the root apical meristem, and glr and glr of arabidopsis were involved in lateral root initiation. all these literature-based evidences support the potential roles of the target genes in rice root development.  summarily, this study on the srnas in the rice root systems could advance our knowledge of the regulatory activities and the biological functions of the plant srnas other than the mirnas. the networks established in this study were supported by the degradome sequencing data, the microarray data and the literature-based evidences, indicating that the networks could provide a basis for in-depth analysis of srna-mediated gene regulation in the rice roots. 
the molecular mechanisms underlying root development have been extensively studied to improve root system architecture, especially for the crops. taken together, the regulatory networks mediated by the srnas highly accumulated in the root tips or in the whole roots could advance our current understanding of the srna-involved molecular mechanisms underlying rice root development. specifically, the expression patterns of certain target genes in the root tips and the whole roots were contrary to those of the regulating srnas. besides, several targets were indicated to play important roles in root development based on literature mining. after argonaute enrichment analysis, the srnas with great potential of performing target cleavages were included for target prediction and degradome sequencing data-based validation. in this study, by using srna high-throughput sequencing data, we made a comparison of the srna accumulation levels between the rice root tips and the whole roots. the srnas highly accumulated in the root tips and in the whole roots were extracted respectively. as a result, lists of the targets regulated by the ago1-enriched srnas were obtained for both the root tips and the whole roots. the root systems play essential roles for plants to anchorage to the soil, and to exploit the mineral and water resources. further evidences were identified from microarray data of the target genes to support some of the srna—target interactions. however, whether the other small rna species, which occupy a dominant portion of the plant endogenous srna population, possess potential roles in root development remains unclear. several microrna families have been demonstrated to be involved in plant root development.
 the root systems play essential roles for plants to anchorage to the soil, and to exploit the mineral and water resources. the molecular mechanisms underlying root development have been extensively studied to improve root system architecture, especially for the crops. several microrna families have been demonstrated to be involved in plant root development. however, whether the other small rna species, which occupy a dominant portion of the plant endogenous srna population, possess potential roles in root development remains unclear.  in this study, by using srna high-throughput sequencing data, we made a comparison of the srna accumulation levels between the rice root tips and the whole roots. the srnas highly accumulated in the root tips and in the whole roots were extracted respectively. after argonaute enrichment analysis, the srnas with great potential of performing target cleavages were included for target prediction and degradome sequencing data-based validation. as a result, lists of the targets regulated by the ago1-enriched srnas were obtained for both the root tips and the whole roots. further evidences were identified from microarray data of the target genes to support some of the srna—target interactions. specifically, the expression patterns of certain target genes in the root tips and the whole roots were contrary to those of the regulating srnas. besides, several targets were indicated to play important roles in root development based on literature mining.  taken together, the regulatory networks mediated by the srnas highly accumulated in the root tips or in the whole roots could advance our current understanding of the srna-involved molecular mechanisms underlying rice root development. and, the srna—target lists could serve as the basis for further functional investigations. small rnawhole rootroot tipargonaute 1degradomenetworkrice 