BACKGROUND
boron -deficiency is a widespread problem in many agricultural crops, including citrus.
over  <dig> crops are susceptible to b-deficiency, and low b availability in soils inhibits vegetative and reproductive growth in a large number of crops  <cit> .
to cope with b-deficiency, plants have evolved a considerable degree of developmental plasticity, including adaptations via cascades of molecular networks.
one of the most obvious features of the adaptations to b-deficiency is the changes in expression profiles of genes involved in a broad spectrum of biochemical, cellular and physiological processes, including b uptake and translocation, carbohydrate and energy metabolism, stress response, signaling and regulation, cell wall, protein process, nucleic acid metabolism, amino acid and fatty acid metabolism  <cit> .
small rnas  have been identified as important post-transcriptional regulators of gene expression in plants.
based on the differences of biogenesis and function, endogenous srnas in plants can been divided into two classes, micrornas  and small interfering rnas .
mirnas, which are approx.
21-nucleotide  in length and are generated from non-coding transcripts capable of forming imperfectly complementary hairpin structures by the rnase dicer-like <dig>  or dcl <dig>  have been known to negatively regulate gene expression at the posttranscriptional level by specific binding and cleavage of their target mrnas, or by repression of target mrna translation  <cit> .
since the first identification of plant mirnas in  <dig>  <cit> , increasing evidence shows that plant mirnas play crucial roles in almost all biological and metabolic processes  <cit> .
therefore, mirna-related research has become one of the hottest topics in plant biology.
in addition to their involvement in plant normal growth and development, mirnas also regulate the adaptations of plants to biotic and abiotic stresses  <cit> .
evidence in arabidopsis thaliana, tomato , rapeseed , rice , and common bean  has demonstrated the important roles of mirnas in phosphorus , nitrogen , sulfur  and cupper  deficiencies  <cit> .
in a. thaliana, mir <dig> has been predicted to target three genes, which encode a phosphate transporter , a ubiquitin-conjugating e <dig> enzyme, and a dead box helicase; however, only the e <dig> enzyme encoded by ubc <dig> has been experimentally validated  <cit> .
mir <dig> is up-regulated in p-deficient roots and suppressed in p-sufficient roots and is negatively correlated with that of its target gene ubc <dig> <cit> .
the inverse relationship of expression patterns between ubc <dig> homologs and mir <dig> under p-deficiency has been confirmed in common bean  <cit>  and rice  <cit> .
transgenic arabidopsis overexpressing mir <dig> also had decreased level of ubc <dig> transcripts  <cit> .
in accordance with being inhibited by mir399s, ubc <dig> down-regulates p uptake and root-to-shoot allocation.
phenotypes of both the arabidopsis t-dna knockout ucb <dig> mutants and the mir399-overexpressing transgenic arabidopsis plants resemble those of a previously reported pho <dig> mutant, a p overaccumulator  <cit> .
therefore, mir <dig> plays important roles in maintaining p homeostasis by regulating ubc <dig> transcript levels  <cit> .
following the first identification, more and more p-deficiency-responsive mirnas are being identified in various plant species, including arabidopsis <cit> , rapeseed  <cit> , soybean   <cit> , white lupin   <cit> , medicago truncatula <cit> , rice  <cit> , switchgrass   <cit> , common bean  <cit>  and tomato  <cit> .
so far, the majority of validated p-deficiency-responsive mirna target genes are transcription factors, and other target genes mainly encode for abiotic/biotic stress-responsive proteins and enzymes related to protein modification/degradation  <cit> .
the diverse functions of these target genes mean that a broad range of biological processes are coordinated in response to p-deficiency.
in a. thaliana, mir <dig> is enhanced during sulfate-limitation, and its induction is controlled by a key transcription factor  in the s assimilation pathway  <cit> .
each plant mirna regulates several genes, but usually the targets belong to the same gene family.
however, mir <dig> targets members of the atp sulfurylase  gene family  <cit>  and the sulfate transporter sultr2; <dig>  <cit> .
mir <dig> has been shown to mediate regulation of sulfate accumulation and allocation by targeting aps and sultr2; <dig>  respectively  <cit> .
recent work showed that in arabidopsis, the expression of mir <dig>  mir <dig>  mir <dig>  and mir <dig> was induced by cu-deficiency and negatively correlated with the accumulation of transcripts for cu:zinc  superoxide dismutase , cox5b- <dig> , plantacyanin and laccases.
it has been suggested that mirna-mediated down-regulation is a general mechanism to regulate non-essential cu proteins, thus allowing plants to save cu for the most essential functions during cu-starvation  <cit> .
also, mirnas have been demonstrated to play important roles in response to n and iron  deficiencies  <cit> .
therefore, mirnas may be involved in the adaptive responses of plant to b-deficiency.
recently, ozhuner et al.
<cit>  investigated b-toxicity-responsive mirnas in barley roots and leaves and concluded that the signal transduction mechanism in leaves regulated by mir <dig> played an important role in barley b-tolerance.
in addition, the expression level of mir <dig> in barley roots and leaves was differentially regulated by b-toxicity.
however, little information about b-deficiency-responsive plant mirnas is available.
identification of mirnas is a key step for understanding their regulatory functions in plants.
plant mirnas were discovered by both experimental and computational approaches.
however, both the computational approach by searching for homologous sequences using est or genomic sequences and the small-scale traditional sequencing approach are mostly limited to the identification of conserved mirnas  <cit> .
recently developed high-throughput sequencing techniques  have become powerful tools to uncover the large list of srna species in plants.
these deep sequencing strategies may identify both known and novel mirnas at unprecedented sensitivities and provide quantitative profiling of mirna expression  <cit> .
citrus belong to evergreen subtropical fruit trees and are commercially grown in many countries.
in  <dig>  morris first described b-deficiency in field grown citrus in south africa  <cit> .
in china, b-deficiency is frequently observed in citrus orchards and is responsible for loss of productivity and poor fruit quality  <cit> .
although the effects of b-deficiency on citrus growth, mineral nutrients, b uptake and distribution, co <dig> assimilation, photosystem ii photochemistry, photosynthetic enzymes, respiration, carbohydrate metabolism, antioxidant system and proteomics have been examined in some details  <cit> , no data are available on b-deficiency-responsive mirnas in citrus.
in this study, we reported the high-throughput sequencing  analysis of srnas from roots of citrus sinensis seedlings grown in b-sufficient  and -deficient nutrient solution with the objectives of identifying mirnas that might be involved in the tolerance of plants to b-deficiency.
RESULTS
plant growth and b concentration in roots and leaves
as shown in table  <dig>  0 μm b treatment decreased shoot and whole plant dry weight , and b concentration in roots and leaves, increased the ratio of root dw to shoot dw, but did not affect root dw.
b concentration in 0 μm b-treated leaves was much lower than the sufficiency range of  <dig> to 100 μg g− <dig> dw  <cit> .
based on these results, plants treated with 0 μm b are considered b-deficient, and those treated with 10 μm b are considered b-sufficient.
b treatments
root dw
shoot dw
whole plant dw
root dw/ shoot dw
b concentration
data are means ± sd from  <dig> to  <dig> replicates.
within a column, values followed by different letters are significantly different at p <  <dig>  detected by the unpaired t-test.
high-throughput sequencing and annotation of mirnas in roots
two libraries were constructed from c. sinensis roots subjected to  <dig> or 10 μm b for 15 weeks, respectively.
these libraries were sequenced by illumina sequencing, leading to the generation of  <dig> , <dig> and  <dig> , <dig> raw reads from libraries of control and b-deficiency, respectively .
after removal of adaptors, low quality tags and contaminants, the length distribution of clean reads was summarized in additional file  <dig>  reads with length of 24 nt were at the most abundant, followed by the reads with length of 22 nt and 21 nt.
b-deficiency resulted in fewer 22 nt and 24 nt reads and more 20 nt and 23 nt reads.
generally speaking, the length distribution of srnas was similar to previous reports in higher plants such as sweet orange   <cit> , medicago truncatula <cit> , arabidopsis <cit>  and trifoliate orange   <cit> .
this suggests that the illumina sequencing data of srna libraries is reliable.
citrus sinensis
as shown in table  <dig>   <dig> , <dig> clean reads  from control and  <dig> , <dig> clean reads  from b-deficient roots were mapped to c. clementina genome using soap.
exon, intron, mirna, rrna, snrna, snorna and trna reads were annotated, respectively.
reads used for prediction of novel mirnas for control and b-deficient roots were  <dig> , <dig> and  <dig> , <dig>  respectively.
identification of known mirnas in roots
to identify the known mirnas in the two libraries constructed from control and b-deficient roots, clear reads were aligned with known plant mirnas from mirbase  <dig> .
only the perfectly matched sequences were considered.
a total of  <dig> known mirnas was identified in the two libraries .
to compare the abundance of mirnas in different libraries, the count of reads was normalized to tpm.
in control library, the most abundant mirna identified was mir <dig> , followed by mir <dig>  and mir <dig> .
however, mir <dig> abundance  in b-deficient library ranked third after mir <dig>  and mir <dig>  .
the known mirnas with normalized read-count less that ten in the two libraries were not used for further analysis, because the use of low expressed mirnas is apt to cause false results  <cit> .
after removing these mirnas, a total of  <dig> mirnas were further analyzed .
identification of novel mirnas in roots
after removal of the rrnas, snrnas, snornas, trnas and known mirnas, the remained sequences that were not annotated were used to predict novel mirnas using the mireap .
based on the criteria for annotation of plant mirnas  <cit> , we identified a total of  <dig> novel mirnas from control and b-deficient roots .
similar to the known mirnas, novel mirnas with very low expression were excluded from the expression analysis  <cit> , thus leading to  <dig> mirnas that were used for further analysis .
differentially expressed mirnas between control and b-deficient roots
a mirna was considered differentially expressed when the mirna had both a fold-change of more that  <dig>  and a p-value of less than  <dig> .
based on the two criteria,  <dig> known and  <dig> novel mirnas were up-regulated, and  <dig> known and  <dig> novel mirnas were down-regulated in response to b-deficiency .
validation of high-throughput sequencing results by real time quantitative reverse transcription pcr
as only one mixed sample of b-deficient and control rna was sequenced, it was necessary to measure the expression of a selection of mirnas to validate that the changes observed were biologically consistent.
qrt-pcr analysis showed that  <dig> of the  <dig> mirnas tested were comparable in magnitude to the expression profiles obtained by the high-throughput sequencing .
this technique was thus validated in  <dig> % of cases.
prediction of targets for differentially expressed mirnas
a total of  <dig>  genes were predicted based on the  <dig>  differentially expressed known  mirnas in c. sinensis roots .
go categories were assigned to all the predicted targets according to the cellular component, molecular function and biological process.
categories based on the cellular component showed that the potential targets for the  <dig> and  <dig> differentially expressed known and novel mirnas were associated with  <dig> and  <dig> components, respectively, with the highest percentage of membrane .
based on the molecular function, the targets for the known and novel mirnas were classified into  <dig> and  <dig> categories, respectively, the highest percentage of two categories were nucleic acid binding and metal ion binding .
as shown in figure 2c, the known and novel mirnas targets were involved in  <dig> and  <dig> biological processes, respectively, the most two go terms are developmental process and response to stress for known mirnas and developmental process and nucleic acid metabolism process for novel mirnas, respectively.
qrt-pcr relative expression analysis of target genes
in plants, genes targeted by mirnas are believed to be regulated mainly via endonucleolytic cleavage of mrnas due to their near-perfect complementarity to their target genes, although evidence indicates the existence of widespread translational inhibition  <cit> .
twenty-nine genes targeted by  <dig> down-regulated and two up-regulated mirnas were assayed by qrt-pcr .
seventeen of the  <dig> target genes had the expected changes in mrna levels, suggesting that mirnas play a role in regulating gene expression under b-deficiency by cleaving mrnas.
however, the expression changes of  <dig> target genes displayed a positive correlation with their corresponding mirnas.
the remaining one target gene was not detected in control and b-deficient roots.
overall, there was no obvious pattern in the expression profiles of target genes in response to b-deficiency.
for example, the relative expression levels of nine genes targeted by down-regulated mir <dig> were validated by qrt-pcr.
four genes displayed decreased expression, while five had increased expression.
the results were consistent with those reported in arabidopsis <cit> .
2+
both fold change of mirnas and relative change of target genes are the ratio of b-deficient roots to the controls.
the value for relative change of target gene is an average of at least three biological replicates with three technical replicates; target genes that had the expected changes in mrna levels were marked in bold.
nd: not detected; *and **indicate a significant difference at p <  <dig>  and p <  <dig> , respectively.
root metabolites and enzymes
b-deficient roots displayed decreased concentration of anthocyanin and increased levels of flavonoids .
b-deficiency increased root proline concentration, and decreased its proline dehydrogenase  activity .
as shown in figure  <dig>  b-deficient roots had increased glutamate dehydrogenase -nad  activity, but decreased gdh-nadh  activity.
discussion
as important post-transcriptional regulators, the expressions of many plant mirnas are regulated by various biotic and abiotic stresses, including nutrient  deficiencies, which may contribute to the development of adaptive responses to deal with unfavorable growth conditions  <cit> .
although the genes responsible for tolerance of plants to b-deficiency has been examined in some detail  <cit> , little information is available on the roles of mirnas under b-deficiency.
in this study, we identified  <dig> known mirnas  and  <dig> novel mirnas  from control and b-deficient roots.
a  <dig> -fold cut-off was set to determine up-regulated and down-regulated mirnas in addition to a p-value of less than  <dig> .
based on the two criteria,  <dig>  up-regulated mirnas and  <dig>  down-regulated mirnas were identified in b-deficient roots , demonstrating that the expression profiles of mirnas in b-deficient roots were greatly affected.
our finding that root mir <dig> was up-regulated under b-deficiency  agrees with the previous results obtained on drought-stressed rice and maize  leaves  <cit>  and salt-stressed maize roots  <cit> .
wei et al.
<cit>  showed that under drought stress, the transcript of mir <dig> and the concentration of proline were increased in maize, whereas its target gene pdh was down-regulated.
they concluded that drought-induced increase in mir <dig> expression might down-regulate pdh, thus increasing the accumulation of proline, an osmoprotectant.
in addition to improving osmoprotection responses to drought stress, proline acts as a free radical scavenger to protect plants from oxidative damage.
hajiboland and bastani  <cit>  observed that proline concentration in tea roots and leaves increased under b-deficiency and drought stress, especially in roots, suggesting that the b-deficiency-induced accumulation of proline may be a strategy for tea plants to counteract the oxidative stress.
therefore, proline level in b-deficient roots might be enhanced due to decreased pdh activity resulting from enhanced expression level of mir <dig>  thus improving the adaptation of plants to b-deficiency.
as expected, b-deficient roots displayed decreased pdh activity and increased proline concentration .
we found that mir <dig> in roots was down-regulated by b-deficiency , as previously obtained on n- and p- deficient common bean roots.
however, common bean root mir <dig> was up-regulated by fe-deficiency and manganese -toxicity  <cit> .
in plants, mir <dig> and mir <dig> have been grouped in one mirna family because of their high degree of sequence similarity and their conserved targets, the squamosa promoter binding protein-like  proteins  <cit> .
gou et al.
<cit>  observed a positive relationship of anthocyanin concentration and mir <dig> activity and an inverse relationship of flavonol  concentration and mir <dig> activity.
as expected, b-deficient roots had lower anthocyanin concentration and higher flavonoid concentration  due to decreased expression of mir <dig>  gou et al.
<cit>  suggested that at one of the mir <dig> targets, spl <dig>  negatively regulates anthocyanin accumulation by directly inhibiting expression of anthocyanin biosynthetic genes through disruption of the myb-bhlh-wd <dig>  however, spl <dig> in c. sinensis roots was down-regulated by b-deficiency .
our observation that root mir <dig> was decreased under b-deficiency  agrees with the previous report that mir <dig> was down-regulated in p-deficient tomato roots  <cit>  and n-deficient arabidopsis seedlings  <cit> .
however, buhtz et al.
<cit>  observed that mir <dig> was up-regulated in the phloem of b. napus under fe-deficiency.
in arabidopsis seedlings, mir <dig> were up-regulated in response to salt stress  <cit> .
expression of mir158a in arabidopsis roots increased under hypoxia  <cit> .
mir <dig> is predicted to target three genes encoding a pentatricopeptide repeat containing protein of unknown function, fucosyltransferases  and a lipase  <cit> .
however, root expression levels of fucosyltransferase  <dig> and lipase class  <dig> family protein decreased in response to b-deficiency .
root transcript of mir <dig> decreased in response to b-deficiency .
this means that its target genes: fkbp-type peptidyl-prolyl cis-trans isomerase family protein and root hair defective  <dig> gtp-binding protein  , might be up-regulated under b-deficiency.
this is validated by our qrt-pcr data that the expression of the two genes in roots increased in response to b-deficiency .
fk506-binding proteins , cyclosporins  and parvulin  are the three major classes of peptidyl prolyl cis-trans isomerases , which are considered to assist chaperones by accelerating the slow rate-limiting isomerization steps.
among these, the best-studied class of ppiases is that of fkbps by far.
increasing evidence shows that in addition to their role in protein folding, plant fkbps are involved in abiotic stress response  <cit> .
nigam et al.
<cit>  showed that yeast cells overexpressing fkbp <dig> displayed enhanced tolerance to high temperatures.
the up-regulation of rhd <dig> in b-deficient roots agrees with the previous results obtained by yang et al.
<cit>  in b-deficient c. sinensis roots and by redondo-nieto et al.
<cit>  in b-deficient m. truncatula root nodules.
in arabidopsis, rhd <dig> has been suggested to be required for cell wall biosynthesis and actin organization  <cit> .
xu et al.
<cit>  observed that transgenic poplar plants overexpressing perhd <dig> had less adventitious roots, more lateral roots, and longer and more root hairs.
thus, the number of lateral roots in b-deficient c. sinensis seedlings might increase due to increased expression level of rhd <dig>  this agrees with the previous reports data that b-deficiency increased lateral root formation of plants  <cit> .
these results imply that rhd <dig> may be involved in the tolerance of plants to b-deficiency.
zhou et al.
<cit>  reported that overexpression of mir <dig> resulted in a drastic reduction in the transcript levels of its target genes  in arabidopsis seedlings.
hawker and bowman  <cit>  showed that hd-zip iii genes played a role in promoting arabidopsis lateral root formation.
therefore, b-deficient roots might have increased expression level of hd-zip iii due to decreased abundance of mir <dig> , thus enhancing lateral root formation.
indeed, qrt-pcr analysis showed that the expression levels of three hd-zip iii transcription factors  increased in b-deficient roots .
this is also supported by the previous reports that b-deficiency increased the lateral root formation  <cit> .
recently, xu et al.
<cit>  observed an inverse relationship between the abundance of mir <dig> in the red-flesh mutant of sweet orange and the expression level of its target gene encoding lycopene β-cyclase, a key enzyme of the carotenoid biosynthesis pathway.
in our study, root mir <dig> was was up-regulated under-b deficiency , implying that carotenoid biosynthesis might be impaired in b-deficient roots due to decreased expression of lycopene β-cyclase gene.
our finding that root mir <dig> was induced by b-deficiency  agrees with the previous reported that the transcript of mir <dig> was enhanced in salt-stressed roots of vigna unguiculata <cit> , nacl, drought and aba treated p. vulgaris seedlings  <cit> , fe-deficient common bean leaves  <cit> , and drought-stressed m. truncatula shoots  <cit> , and with our qrt-pcr data that the expression of one target gene encoding lrr and nb-arc domains-containing disease resistance protein decreased in b-deficient roots .
however, p-deficiency and mn-toxicity down-regulated the transcript of mir <dig> in common bean leaves  <cit> .
wong et al.
<cit>  reported that the abundance of disease resistance protein  mrna in thellungiella salsuginea leaves was down-regulated in response to drought and short-term salinity stress.
however, the abundance of tir-nbs-lrr class disease resistance protein in thellungiella halophila leaves increased under long-term salinity stress  <cit> .
therefore, mir <dig> might be involved in abiotic stresses as well as biotic stresses.
evidence shows that the target genes of mir <dig> are involved in disease resistance  <cit> .
we found that mir <dig> was enhanced in b-deficient roots , meaning that disease resistance protein gene might be down-regulated, thus decreasing the disease resistance of roots.
as expected, the expression of three genes encoding disease resistance protein  family, lrr and nb-arc domains-containing disease resistance protein, and disease resistance protein  family decreased in b-deficient roots .
this agrees with the fact that b increases the disease resistance in plants  <cit> .
we found that root mir <dig> was up-regulated by b-deficiency , as previously reported on p-starved tomato roots  <cit> , nacl-treated arabidopsis seedlings  <cit> , fe-deficient malus xiaojinensis roots  <cit> , and n-deficient maize shoots   <cit> .
ni et al.
<cit>  showed that overexpression of mir394a in arabidopsis reduced the transcript of an f-box gene  containing a mir <dig> complementary target site.
in arabidopsis, a null mutation in dor gene, which encodes a putative f-box protein, led to a substantial increase in drought tolerance as well as a hypersensitive aba response of stomatal closing; conversely, the transgenic plants overexpressing dor gene showed decreased drought tolerance  <cit> .
song et al.
<cit>  reported that both mir <dig> and lcr transcripts were regulated by salt and drought stresses and aba treatment, concluding that the silencing of lcr mrna by mir <dig> is essential to maintain a certain phenotype favorable for the adaptive response to abiotic stresses.
therefore, b-deficiency might down-regulate the accumulation of lcr mrna in c. sinensis roots due to increased transcript of mir <dig> , thus improving the tolerance of plants to b-deficiency.
however, li et al.
<cit>  observed that mir394a was up-regulated in response to drought stress but down-regulated in response to salinity stress in soybean roots.
mir394a,b,c were up-regulated in roots, stems and leaves of b. napus by sulfate-deficiency and cd stress except for the down-regulation of mir <dig> in sulfate-deficient leaves  <cit> .
in arabidopsis, root mir394b and shoot mir394a and mir394b were initially up-regulated and then down-regulated under fe-deficiency  <cit> .
mir <dig> is predicted to target genes encoding maize protein disulfide isomerase   <cit>  and myb transcription factor   <cit> .
our result showed that root expression of mir <dig> decreased in response to b-deficiency , implying that the expression of pdi and mybml <dig> might be up-regulated in b-deficient roots.
protein disulfide isomerases  are molecular chaperones that contain thioredoxin  domains and aid in the formation of proper disulfide bonds during protein folding.
chen et al.
<cit>  showed that transgenic rice seedlings overexpressing a protein disulfide isomerase-like protein  gene displayed enhanced tolerance to mercury , accompanied by lower levels of superoxide anion radicals, h2o <dig> and malondialdehyde , higher activities of superoxide dismutase  and peroxidase , and increased concentrations of non-protein thiols and reduced glutathione .
plant myb proteins are characterized by a highly conserved myb dna-binding domain.
plant myb transcription factors are involved in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses.
rubio et al.
<cit>  showed that a conserved myb transcription factor was involved in phosphate starvation signaling in both vascular plants and in unicellular algae.
thus, the down-regulation of mir <dig> in b-deficient roots might provide an adaptive strategy of plants to b-deficiency.
b-deficiency decreased the transcript of root mir <dig> .
this agrees with the previous report that mir830a in arabidopsis seedlings was down-regulated at low temperature   <cit> .
however, mir <dig> was up-regulated in p-deficient roots and down-regulated in p-deficient stems and leaves of white lupin  <cit> .
mir <dig> is predicted to target two genes encoding ranbp <dig> domain-containing protein and kinesin motor-related  <cit> .
qrt-pcr analysis showed that b-deficient roots had increased expression of kinesin motor-related, but decreased expression of ranbp <dig> domain-containing protein .
kinesins, a superfamily of microtubule motor proteins ubiquitous in all eukaryotic organisms, function in the unidirectional transport of vesicles and organelles, cytokinesis, signal transduction, and morphogenesis  <cit> .
therefore, the down-regulation of mir <dig> in b-deficient roots might be advantageous to normal growth and development of plants under b-deficiency.
we found that the transcript of mir <dig> decreased in b-deficient roots , implying that its target genes: leucine rich repeat protein , plant homedomain -finger, oligomeric golgi complex 7-like <cit>  and sulfate transporter <cit> , might be up-regulated under b-deficiency.
qrt-pcr analysis showed that b-deficient roots had enhanced expression of lrp, but decreased expression of sulfate transporter .
park et al.
<cit>  observed that heterologous expression of rice lrp  resulted in the activation of defense response and enhanced resistance to bacterial soft rot in chinese cabbage.
thus, the higher expression of lrp might partially compensate for the decreased disease resistance in b-deficient plants  <cit> .
wei et al.
<cit>  reported that transgenic arabidopsis plants overexpressing the gmphd <dig> from soybean displayed enhanced salt tolerance through control of ros signaling and scavenging.
liu et al.
<cit>  observed that overexpression of osphd <dig> enhanced the tolerance of transgenic rice plants to drought, high salt and cold stresses.
therefore, the down-regulation of mir <dig> in b-deficient roots might be an adaptive response.
our observation that b-deficiency decreased the transcript of root sulfate transporter  disagrees with the previous reports that b-deficiency increased the abundances of phosphate transporter 3; <dig>  <cit>  in c. sinensis roots and of k+ channel in b. napus roots  <cit> .
we observed that b-deficiency increased the transcript of mir <dig> in roots , which agrees with the previous data that mir <dig> was expressed in roots of salt-stressed plants, and not expressed in healthy, non-stressed plants  <cit> .
down-regulation of its target gene, putative enoyl-coa hydratase/isomerase by mir <dig> is indicative of the impact of the β-oxidation pathway of unsaturated fatty acids, which might lead to decrease in carbon flux in the form of acetyl-coa.
the acetyl-coa can eventually enter the tca cycle  <cit> .
this agrees with our report that b-deficient c. sinensis roots displayed decreased root respiration  <cit> .
in this study, the transcript of gdh <dig>  a target gene of mir <dig>  <cit> , might be down-regulated in b-deficient roots, thus decreasing the activity of root gdh.
as expected, gdh-nadh  activity was down-regulated in b-deficient c. sinensis roots .
however, gdh-nad  activity in roots increased in response to b-deficiency .
robinson et al.
<cit>  reported that the primary role of gdh was the oxidation of glutamate under conditions where carbon is limited.
thus, b-deficiency-induced increase in gdh-nad activity agrees with the previous study showing that b-deficiency decreased or did not affect root concentrations of non-structural carbohydrates  <cit> .
beato et al.
<cit>  showed that gdh genes, ntgdh-nad;a <dig> and ntgdh-nad;b <dig>  were up-regulated in n-deficient tobacco roots, accompanied by decreased concentrations of glucose and fructose.
b-deficiency decreased the expression level of root mir <dig> .
as expected, the transcript of ammonium transporter 1; <dig> targeted by mir <dig>  was enhanced in b-deficient roots , hence facilitating the uptake of ammonium from external environments as well as the translocation of ammonium from roots to shoots  <cit> .
this agrees with the previous reports that b-deficient tobacco roots had higher concentration of ammonium  <cit> .
the higher uptake of ammonium might compensate for the reduced nitrate uptake by repressing root plasmalemma h+-atpase  gene expression  <cit> .
however, the expression of ammonium transporter  <dig> targeted by mir <dig>  decreased in b-deficient roots .
the expression of autoinhibited ca2+-atpase  <dig> gene , might be up-regulated in b-deficient roots due to decreased transcript of mir <dig> .
this agrees with our finding that b-deficiency increased the abundances of autoinhibited ca2+-atpase  <dig> in c. sinensis roots  <cit> .
CONCLUSIONS
we first identified mirnas and their expression pattern in b-deficient c. sinensis roots by illumina sequencing.
a total of  <dig> known mirnas and of  <dig> novel mirnas was identified from control and b-deficient roots.
in b-deficient roots,  <dig>  up-regulated and  <dig>  down-regulated mirnas were isolated.
this demonstrates remarkable metabolic flexibility of c. sinensis roots, which might contribute to the tolerance of roots to b-deficiency.
a model for the possible roles of mirnas in the tolerance of roots to b-deficiency was proposed through the integration of the present results and available data in the literature .
mirnas might regulate the adaptations of c. sinensis roots to b-deficiency through following several aspects:  activation of the defense response, ros signaling and scavenging due to increased expression of mir <dig> and decreased expression of mir <dig> and mir843;  increasing the number of lateral roots  and maintaining a certain phenotype favorable for the adaptive response to b-deficiency ;  enhancing cell transport by decreasing the accumulation of mir <dig>  mir <dig> and mir3465;  improving osmoprotection  and regulating other metabolic reactions .
in addition, both mir <dig> and mir <dig> expression increased in b-deficient c. sinensis roots, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots.
therefore, the discovery and characterization of these b-deficiency-responsive mirnas will help us to elucidate the molecular mechanisms involved in the tolerance of plants to b-deficiency.
although the absolute conditions without b created under pot conditions do not exist in field, because there is always certain level of b supply in field conditions even under highly b-deficient conditions, pot results should stand ture under field conditions, because typical b-deficient symptoms: corky split veins of citrus leaves usually occur in the sand culture and in field conditions  <cit> .
citrus sinensis
roots to b-deficiency.

 boron -deficiency is a widespread problem in many agricultural crops, including citrus. over crops are susceptible to b-deficiency, and low b availability in soils inhibits vegetative and reproductive growth in a large number of crops. to cope with b-deficiency, plants have evolved a considerable degree of developmental plasticity, including adaptations via cascades of molecular networks. one of the most obvious features of the adaptations to b-deficiency is the changes in expression profiles of genes involved in a broad spectrum of biochemical, cellular and physiological processes, including b uptake and translocation, carbohydrate and energy metabolism, stress response, signaling and regulation, cell wall, protein process, nucleic acid metabolism, amino acid and fatty acid metabolism. small rnas have been identified as important post-transcriptional regulators of gene expression in plants. based on the differences of biogenesis and function, endogenous srnas in plants can been divided into two classes, micrornas and small interfering rnas. mirnas, which are approx. 21-nucleotide in length and are generated from non-coding transcripts capable of forming imperfectly complementary hairpin structures by the rnase dicer-like or dcl have been known to negatively regulate gene expression at the posttranscriptional level by specific binding and cleavage of their target mrnas, or by repression of target mrna translation. since the first identification of plant mirnas in, increasing evidence shows that plant mirnas play crucial roles in almost all biological and metabolic processes. therefore, mirna-related research has become one of the hottest topics in plant biology. in addition to their involvement in plant normal growth and development, mirnas also regulate the adaptations of plants to biotic and abiotic stresses. evidence in arabidopsis thaliana, tomato, rapeseed, rice, and common bean has demonstrated the important roles of mirnas in phosphorus, nitrogen, sulfur and cupper deficiencies. in a. thaliana, mir has been predicted to target three genes, which encode a phosphate transporter, a ubiquitin-conjugating e enzyme, and a dead box helicase; however, only the e enzyme encoded by ubc has been experimentally validated. mir is up-regulated in p-deficient roots and suppressed in p-sufficient roots and is negatively correlated with that of its target gene ubc. the inverse relationship of expression patterns between ubc homologs and mir under p-deficiency has been confirmed in common bean and rice. transgenic arabidopsis overexpressing mir also had decreased level of ubc transcripts. in accordance with being inhibited by mir399s, ubc down-regulates p uptake and root-to-shoot allocation. phenotypes of both the arabidopsis t-dna knockout ucb mutants and the mir399-overexpressing transgenic arabidopsis plants resemble those of a previously reported pho mutant, a p overaccumulator. therefore, mir plays important roles in maintaining p homeostasis by regulating ubc transcript levels. following the first identification, more and more p-deficiency-responsive mirnas are being identified in various plant species, including arabidopsis, rapeseed, soybean, white lupin, medicago truncatula, rice, switchgrass, common bean and tomato. so far, the majority of validated p-deficiency-responsive mirna target genes are transcription factors, and other target genes mainly encode for abiotic/biotic stress-responsive proteins and enzymes related to protein modification/degradation. the diverse functions of these target genes mean that a broad range of biological processes are coordinated in response to p-deficiency. in a. thaliana, mir is enhanced during sulfate-limitation, and its induction is controlled by a key transcription factor in the s assimilation pathway. each plant mirna regulates several genes, but usually the targets belong to the same gene family. however, mir targets members of the atp sulfurylase gene family and the sulfate transporter sultr2;. mir has been shown to mediate regulation of sulfate accumulation and allocation by targeting aps and sultr2; respectively. recent work showed that in arabidopsis, the expression of mir mir mir and mir was induced by cu-deficiency and negatively correlated with the accumulation of transcripts for cu:zinc superoxide dismutase, cox5b-, plantacyanin and laccases. it has been suggested that mirna-mediated down-regulation is a general mechanism to regulate non-essential cu proteins, thus allowing plants to save cu for the most essential functions during cu-starvation. also, mirnas have been demonstrated to play important roles in response to n and iron deficiencies. therefore, mirnas may be involved in the adaptive responses of plant to b-deficiency. recently, ozhuner et al. investigated b-toxicity-responsive mirnas in barley roots and leaves and concluded that the signal transduction mechanism in leaves regulated by mir played an important role in barley b-tolerance. in addition, the expression level of mir in barley roots and leaves was differentially regulated by b-toxicity. however, little information about b-deficiency-responsive plant mirnas is available. identification of mirnas is a key step for understanding their regulatory functions in plants. plant mirnas were discovered by both experimental and computational approaches. however, both the computational approach by searching for homologous sequences using est or genomic sequences and the small-scale traditional sequencing approach are mostly limited to the identification of conserved mirnas. recently developed high-throughput sequencing techniques have become powerful tools to uncover the large list of srna species in plants. these deep sequencing strategies may identify both known and novel mirnas at unprecedented sensitivities and provide quantitative profiling of mirna expression. citrus belong to evergreen subtropical fruit trees and are commercially grown in many countries. in morris first described b-deficiency in field grown citrus in south africa. in china, b-deficiency is frequently observed in citrus orchards and is responsible for loss of productivity and poor fruit quality. although the effects of b-deficiency on citrus growth, mineral nutrients, b uptake and distribution, co assimilation, photosystem ii photochemistry, photosynthetic enzymes, respiration, carbohydrate metabolism, antioxidant system and proteomics have been examined in some details, no data are available on b-deficiency-responsive mirnas in citrus. in this study, we reported the high-throughput sequencing analysis of srnas from roots of citrus sinensis seedlings grown in b-sufficient and -deficient nutrient solution with the objectives of identifying mirnas that might be involved in the tolerance of plants to b-deficiency.  plant growth and b concentration in roots and leaves as shown in table  0 μm b treatment decreased shoot and whole plant dry weight, and b concentration in roots and leaves, increased the ratio of root dw to shoot dw, but did not affect root dw. b concentration in 0 μm b-treated leaves was much lower than the sufficiency range of to 100 μg g− dw. based on these results, plants treated with 0 μm b are considered b-deficient, and those treated with 10 μm b are considered b-sufficient. b treatments root dw shoot dw whole plant dw root dw/ shoot dw b concentration data are means ± sd from to replicates. within a column, values followed by different letters are significantly different at p <  detected by the unpaired t-test. high-throughput sequencing and annotation of mirnas in roots two libraries were constructed from c. sinensis roots subjected to or 10 μm b for 15 weeks, respectively. these libraries were sequenced by illumina sequencing, leading to the generation of, and, raw reads from libraries of control and b-deficiency, respectively. after removal of adaptors, low quality tags and contaminants, the length distribution of clean reads was summarized in additional file reads with length of 24 nt were at the most abundant, followed by the reads with length of 22 nt and 21 nt. b-deficiency resulted in fewer 22 nt and 24 nt reads and more 20 nt and 23 nt reads. generally speaking, the length distribution of srnas was similar to previous reports in higher plants such as sweet orange, medicago truncatula, arabidopsis and trifoliate orange. this suggests that the illumina sequencing data of srna libraries is reliable. citrus sinensis as shown in table , clean reads from control and, clean reads from b-deficient roots were mapped to c. clementina genome using soap. exon, intron, mirna, rrna, snrna, snorna and trna reads were annotated, respectively. reads used for prediction of novel mirnas for control and b-deficient roots were, and, respectively. identification of known mirnas in roots to identify the known mirnas in the two libraries constructed from control and b-deficient roots, clear reads were aligned with known plant mirnas from mirbase. only the perfectly matched sequences were considered. a total of known mirnas was identified in the two libraries. to compare the abundance of mirnas in different libraries, the count of reads was normalized to tpm. in control library, the most abundant mirna identified was mir, followed by mir and mir. however, mir abundance in b-deficient library ranked third after mir and mir. the known mirnas with normalized read-count less that ten in the two libraries were not used for further analysis, because the use of low expressed mirnas is apt to cause false results. after removing these mirnas, a total of mirnas were further analyzed. identification of novel mirnas in roots after removal of the rrnas, snrnas, snornas, trnas and known mirnas, the remained sequences that were not annotated were used to predict novel mirnas using the mireap. based on the criteria for annotation of plant mirnas, we identified a total of novel mirnas from control and b-deficient roots. similar to the known mirnas, novel mirnas with very low expression were excluded from the expression analysis, thus leading to mirnas that were used for further analysis. differentially expressed mirnas between control and b-deficient roots a mirna was considered differentially expressed when the mirna had both a fold-change of more that and a p-value of less than. based on the two criteria, known and novel mirnas were up-regulated, and known and novel mirnas were down-regulated in response to b-deficiency. validation of high-throughput sequencing results by real time quantitative reverse transcription pcr as only one mixed sample of b-deficient and control rna was sequenced, it was necessary to measure the expression of a selection of mirnas to validate that the changes observed were biologically consistent. qrt-pcr analysis showed that of the mirnas tested were comparable in magnitude to the expression profiles obtained by the high-throughput sequencing. this technique was thus validated in % of cases. prediction of targets for differentially expressed mirnas a total of genes were predicted based on the differentially expressed known mirnas in c. sinensis roots. go categories were assigned to all the predicted targets according to the cellular component, molecular function and biological process. categories based on the cellular component showed that the potential targets for the and differentially expressed known and novel mirnas were associated with and components, respectively, with the highest percentage of membrane. based on the molecular function, the targets for the known and novel mirnas were classified into and categories, respectively, the highest percentage of two categories were nucleic acid binding and metal ion binding. as shown in figure 2c, the known and novel mirnas targets were involved in and biological processes, respectively, the most two go terms are developmental process and response to stress for known mirnas and developmental process and nucleic acid metabolism process for novel mirnas, respectively. qrt-pcr relative expression analysis of target genes in plants, genes targeted by mirnas are believed to be regulated mainly via endonucleolytic cleavage of mrnas due to their near-perfect complementarity to their target genes, although evidence indicates the existence of widespread translational inhibition. twenty-nine genes targeted by down-regulated and two up-regulated mirnas were assayed by qrt-pcr. seventeen of the target genes had the expected changes in mrna levels, suggesting that mirnas play a role in regulating gene expression under b-deficiency by cleaving mrnas. however, the expression changes of target genes displayed a positive correlation with their corresponding mirnas. the remaining one target gene was not detected in control and b-deficient roots. overall, there was no obvious pattern in the expression profiles of target genes in response to b-deficiency. for example, the relative expression levels of nine genes targeted by down-regulated mir were validated by qrt-pcr. four genes displayed decreased expression, while five had increased expression. the results were consistent with those reported in arabidopsis. 2+ both fold change of mirnas and relative change of target genes are the ratio of b-deficient roots to the controls. the value for relative change of target gene is an average of at least three biological replicates with three technical replicates; target genes that had the expected changes in mrna levels were marked in bold. nd: not detected; *and **indicate a significant difference at p <  and p < , respectively. root metabolites and enzymes b-deficient roots displayed decreased concentration of anthocyanin and increased levels of flavonoids. b-deficiency increased root proline concentration, and decreased its proline dehydrogenase activity. as shown in figure  b-deficient roots had increased glutamate dehydrogenase -nad activity, but decreased gdh-nadh activity. discussion as important post-transcriptional regulators, the expressions of many plant mirnas are regulated by various biotic and abiotic stresses, including nutrient deficiencies, which may contribute to the development of adaptive responses to deal with unfavorable growth conditions. although the genes responsible for tolerance of plants to b-deficiency has been examined in some detail, little information is available on the roles of mirnas under b-deficiency. in this study, we identified known mirnas and novel mirnas from control and b-deficient roots. a -fold cut-off was set to determine up-regulated and down-regulated mirnas in addition to a p-value of less than. based on the two criteria, up-regulated mirnas and down-regulated mirnas were identified in b-deficient roots, demonstrating that the expression profiles of mirnas in b-deficient roots were greatly affected. our finding that root mir was up-regulated under b-deficiency agrees with the previous results obtained on drought-stressed rice and maize leaves and salt-stressed maize roots. wei et al. showed that under drought stress, the transcript of mir and the concentration of proline were increased in maize, whereas its target gene pdh was down-regulated. they concluded that drought-induced increase in mir expression might down-regulate pdh, thus increasing the accumulation of proline, an osmoprotectant. in addition to improving osmoprotection responses to drought stress, proline acts as a free radical scavenger to protect plants from oxidative damage. hajiboland and bastani observed that proline concentration in tea roots and leaves increased under b-deficiency and drought stress, especially in roots, suggesting that the b-deficiency-induced accumulation of proline may be a strategy for tea plants to counteract the oxidative stress. therefore, proline level in b-deficient roots might be enhanced due to decreased pdh activity resulting from enhanced expression level of mir thus improving the adaptation of plants to b-deficiency. as expected, b-deficient roots displayed decreased pdh activity and increased proline concentration. we found that mir in roots was down-regulated by b-deficiency, as previously obtained on n- and p- deficient common bean roots. however, common bean root mir was up-regulated by fe-deficiency and manganese -toxicity. in plants, mir and mir have been grouped in one mirna family because of their high degree of sequence similarity and their conserved targets, the squamosa promoter binding protein-like proteins. gou et al. observed a positive relationship of anthocyanin concentration and mir activity and an inverse relationship of flavonol concentration and mir activity. as expected, b-deficient roots had lower anthocyanin concentration and higher flavonoid concentration due to decreased expression of mir gou et al. suggested that at one of the mir targets, spl negatively regulates anthocyanin accumulation by directly inhibiting expression of anthocyanin biosynthetic genes through disruption of the myb-bhlh-wd however, spl in c. sinensis roots was down-regulated by b-deficiency. our observation that root mir was decreased under b-deficiency agrees with the previous report that mir was down-regulated in p-deficient tomato roots and n-deficient arabidopsis seedlings. however, buhtz et al. observed that mir was up-regulated in the phloem of b. napus under fe-deficiency. in arabidopsis seedlings, mir were up-regulated in response to salt stress. expression of mir158a in arabidopsis roots increased under hypoxia. mir is predicted to target three genes encoding a pentatricopeptide repeat containing protein of unknown function, fucosyltransferases and a lipase. however, root expression levels of fucosyltransferase and lipase class family protein decreased in response to b-deficiency. root transcript of mir decreased in response to b-deficiency. this means that its target genes: fkbp-type peptidyl-prolyl cis-trans isomerase family protein and root hair defective gtp-binding protein, might be up-regulated under b-deficiency. this is validated by our qrt-pcr data that the expression of the two genes in roots increased in response to b-deficiency. fk506-binding proteins, cyclosporins and parvulin are the three major classes of peptidyl prolyl cis-trans isomerases, which are considered to assist chaperones by accelerating the slow rate-limiting isomerization steps. among these, the best-studied class of ppiases is that of fkbps by far. increasing evidence shows that in addition to their role in protein folding, plant fkbps are involved in abiotic stress response. nigam et al. showed that yeast cells overexpressing fkbp displayed enhanced tolerance to high temperatures. the up-regulation of rhd in b-deficient roots agrees with the previous results obtained by yang et al. in b-deficient c. sinensis roots and by redondo-nieto et al. in b-deficient m. truncatula root nodules. in arabidopsis, rhd has been suggested to be required for cell wall biosynthesis and actin organization. xu et al. observed that transgenic poplar plants overexpressing perhd had less adventitious roots, more lateral roots, and longer and more root hairs. thus, the number of lateral roots in b-deficient c. sinensis seedlings might increase due to increased expression level of rhd this agrees with the previous reports data that b-deficiency increased lateral root formation of plants. these results imply that rhd may be involved in the tolerance of plants to b-deficiency. zhou et al. reported that overexpression of mir resulted in a drastic reduction in the transcript levels of its target genes in arabidopsis seedlings. hawker and bowman showed that hd-zip iii genes played a role in promoting arabidopsis lateral root formation. therefore, b-deficient roots might have increased expression level of hd-zip iii due to decreased abundance of mir, thus enhancing lateral root formation. indeed, qrt-pcr analysis showed that the expression levels of three hd-zip iii transcription factors increased in b-deficient roots. this is also supported by the previous reports that b-deficiency increased the lateral root formation. recently, xu et al. observed an inverse relationship between the abundance of mir in the red-flesh mutant of sweet orange and the expression level of its target gene encoding lycopene β-cyclase, a key enzyme of the carotenoid biosynthesis pathway. in our study, root mir was was up-regulated under-b deficiency, implying that carotenoid biosynthesis might be impaired in b-deficient roots due to decreased expression of lycopene β-cyclase gene. our finding that root mir was induced by b-deficiency agrees with the previous reported that the transcript of mir was enhanced in salt-stressed roots of vigna unguiculata, nacl, drought and aba treated p. vulgaris seedlings, fe-deficient common bean leaves, and drought-stressed m. truncatula shoots, and with our qrt-pcr data that the expression of one target gene encoding lrr and nb-arc domains-containing disease resistance protein decreased in b-deficient roots. however, p-deficiency and mn-toxicity down-regulated the transcript of mir in common bean leaves. wong et al. reported that the abundance of disease resistance protein mrna in thellungiella salsuginea leaves was down-regulated in response to drought and short-term salinity stress. however, the abundance of tir-nbs-lrr class disease resistance protein in thellungiella halophila leaves increased under long-term salinity stress. therefore, mir might be involved in abiotic stresses as well as biotic stresses. evidence shows that the target genes of mir are involved in disease resistance. we found that mir was enhanced in b-deficient roots, meaning that disease resistance protein gene might be down-regulated, thus decreasing the disease resistance of roots. as expected, the expression of three genes encoding disease resistance protein family, lrr and nb-arc domains-containing disease resistance protein, and disease resistance protein family decreased in b-deficient roots. this agrees with the fact that b increases the disease resistance in plants. we found that root mir was up-regulated by b-deficiency, as previously reported on p-starved tomato roots, nacl-treated arabidopsis seedlings, fe-deficient malus xiaojinensis roots, and n-deficient maize shoots. ni et al. showed that overexpression of mir394a in arabidopsis reduced the transcript of an f-box gene containing a mir complementary target site. in arabidopsis, a null mutation in dor gene, which encodes a putative f-box protein, led to a substantial increase in drought tolerance as well as a hypersensitive aba response of stomatal closing; conversely, the transgenic plants overexpressing dor gene showed decreased drought tolerance. song et al. reported that both mir and lcr transcripts were regulated by salt and drought stresses and aba treatment, concluding that the silencing of lcr mrna by mir is essential to maintain a certain phenotype favorable for the adaptive response to abiotic stresses. therefore, b-deficiency might down-regulate the accumulation of lcr mrna in c. sinensis roots due to increased transcript of mir, thus improving the tolerance of plants to b-deficiency. however, li et al. observed that mir394a was up-regulated in response to drought stress but down-regulated in response to salinity stress in soybean roots. mir394a,b,c were up-regulated in roots, stems and leaves of b. napus by sulfate-deficiency and cd stress except for the down-regulation of mir in sulfate-deficient leaves. in arabidopsis, root mir394b and shoot mir394a and mir394b were initially up-regulated and then down-regulated under fe-deficiency. mir is predicted to target genes encoding maize protein disulfide isomerase and myb transcription factor. our result showed that root expression of mir decreased in response to b-deficiency, implying that the expression of pdi and mybml might be up-regulated in b-deficient roots. protein disulfide isomerases are molecular chaperones that contain thioredoxin domains and aid in the formation of proper disulfide bonds during protein folding. chen et al. showed that transgenic rice seedlings overexpressing a protein disulfide isomerase-like protein gene displayed enhanced tolerance to mercury, accompanied by lower levels of superoxide anion radicals, h2o and malondialdehyde, higher activities of superoxide dismutase and peroxidase, and increased concentrations of non-protein thiols and reduced glutathione. plant myb proteins are characterized by a highly conserved myb dna-binding domain. plant myb transcription factors are involved in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. rubio et al. showed that a conserved myb transcription factor was involved in phosphate starvation signaling in both vascular plants and in unicellular algae. thus, the down-regulation of mir in b-deficient roots might provide an adaptive strategy of plants to b-deficiency. b-deficiency decreased the transcript of root mir. this agrees with the previous report that mir830a in arabidopsis seedlings was down-regulated at low temperature. however, mir was up-regulated in p-deficient roots and down-regulated in p-deficient stems and leaves of white lupin. mir is predicted to target two genes encoding ranbp domain-containing protein and kinesin motor-related. qrt-pcr analysis showed that b-deficient roots had increased expression of kinesin motor-related, but decreased expression of ranbp domain-containing protein. kinesins, a superfamily of microtubule motor proteins ubiquitous in all eukaryotic organisms, function in the unidirectional transport of vesicles and organelles, cytokinesis, signal transduction, and morphogenesis. therefore, the down-regulation of mir in b-deficient roots might be advantageous to normal growth and development of plants under b-deficiency. we found that the transcript of mir decreased in b-deficient roots, implying that its target genes: leucine rich repeat protein, plant homedomain -finger, oligomeric golgi complex 7-like and sulfate transporter, might be up-regulated under b-deficiency. qrt-pcr analysis showed that b-deficient roots had enhanced expression of lrp, but decreased expression of sulfate transporter. park et al. observed that heterologous expression of rice lrp resulted in the activation of defense response and enhanced resistance to bacterial soft rot in chinese cabbage. thus, the higher expression of lrp might partially compensate for the decreased disease resistance in b-deficient plants. wei et al. reported that transgenic arabidopsis plants overexpressing the gmphd from soybean displayed enhanced salt tolerance through control of ros signaling and scavenging. liu et al. observed that overexpression of osphd enhanced the tolerance of transgenic rice plants to drought, high salt and cold stresses. therefore, the down-regulation of mir in b-deficient roots might be an adaptive response. our observation that b-deficiency decreased the transcript of root sulfate transporter disagrees with the previous reports that b-deficiency increased the abundances of phosphate transporter 3; in c. sinensis roots and of k+ channel in b. napus roots. we observed that b-deficiency increased the transcript of mir in roots, which agrees with the previous data that mir was expressed in roots of salt-stressed plants, and not expressed in healthy, non-stressed plants. down-regulation of its target gene, putative enoyl-coa hydratase/isomerase by mir is indicative of the impact of the β-oxidation pathway of unsaturated fatty acids, which might lead to decrease in carbon flux in the form of acetyl-coa. the acetyl-coa can eventually enter the tca cycle. this agrees with our report that b-deficient c. sinensis roots displayed decreased root respiration. in this study, the transcript of gdh a target gene of mir, might be down-regulated in b-deficient roots, thus decreasing the activity of root gdh. as expected, gdh-nadh activity was down-regulated in b-deficient c. sinensis roots. however, gdh-nad activity in roots increased in response to b-deficiency. robinson et al. reported that the primary role of gdh was the oxidation of glutamate under conditions where carbon is limited. thus, b-deficiency-induced increase in gdh-nad activity agrees with the previous study showing that b-deficiency decreased or did not affect root concentrations of non-structural carbohydrates. beato et al. showed that gdh genes, ntgdh-nad;a and ntgdh-nad;b were up-regulated in n-deficient tobacco roots, accompanied by decreased concentrations of glucose and fructose. b-deficiency decreased the expression level of root mir. as expected, the transcript of ammonium transporter 1; targeted by mir was enhanced in b-deficient roots, hence facilitating the uptake of ammonium from external environments as well as the translocation of ammonium from roots to shoots. this agrees with the previous reports that b-deficient tobacco roots had higher concentration of ammonium. the higher uptake of ammonium might compensate for the reduced nitrate uptake by repressing root plasmalemma h+-atpase gene expression. however, the expression of ammonium transporter targeted by mir decreased in b-deficient roots. the expression of autoinhibited ca2+-atpase gene, might be up-regulated in b-deficient roots due to decreased transcript of mir. this agrees with our finding that b-deficiency increased the abundances of autoinhibited ca2+-atpase in c. sinensis roots.  we first identified mirnas and their expression pattern in b-deficient c. sinensis roots by illumina sequencing. a total of known mirnas and of novel mirnas was identified from control and b-deficient roots. in b-deficient roots, up-regulated and down-regulated mirnas were isolated. this demonstrates remarkable metabolic flexibility of c. sinensis roots, which might contribute to the tolerance of roots to b-deficiency. a model for the possible roles of mirnas in the tolerance of roots to b-deficiency was proposed through the integration of the present results and available data in the literature. mirnas might regulate the adaptations of c. sinensis roots to b-deficiency through following several aspects: activation of the defense response, ros signaling and scavenging due to increased expression of mir and decreased expression of mir and mir843; increasing the number of lateral roots and maintaining a certain phenotype favorable for the adaptive response to b-deficiency; enhancing cell transport by decreasing the accumulation of mir mir and mir3465; improving osmoprotection and regulating other metabolic reactions. in addition, both mir and mir expression increased in b-deficient c. sinensis roots, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots. therefore, the discovery and characterization of these b-deficiency-responsive mirnas will help us to elucidate the molecular mechanisms involved in the tolerance of plants to b-deficiency. although the absolute conditions without b created under pot conditions do not exist in field, because there is always certain level of b supply in field conditions even under highly b-deficient conditions, pot results should stand ture under field conditions, because typical b-deficient symptoms: corky split veins of citrus leaves usually occur in the sand culture and in field conditions. citrus sinensis roots to b-deficiency. 
however, little is known on b-deficiency-responsive mirnas in plants. we isolated up-regulated and down-regulated mirnas from b-deficient roots, demonstrating remarkable metabolic flexibility of roots, which might contribute to the tolerance of plants to b-deficiency. other mirnas such as mir and mir in roots increased in response to b-deficiency, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots. mirnas might regulate the adaptations of roots to b-deficiency through following several aspects: inactivating reactive oxygen species signaling and scavenging through up-regulating mir and down-regulating mir and mir843; increasing lateral root number by lowering mir expression and maintaining a certain phenotype favorable for b-deficiency-tolerance by increasing mir expression; enhancing cell transport by decreasing the transcripts of mir mir and mir3465; improving osmoprotection and regulating other metabolic reactions. micrornas play important roles in nutrient deficiencies. boron -deficiency is a widespread problem in many crops, including citrus. a model for the possible roles of mirnas in the tolerance of roots to b-deficiency was proposed. our work demonstrates the possible roles of mirnas and related mechanisms in the response of plant roots to b-deficiency. in this study, we first identified mirnas and their expression pattern in b-deficient citrus sinensis roots by illumina sequencing in order to identify mirnas that might be involved in the tolerance of plants to b-deficiency.
 boron -deficiency is a widespread problem in many crops, including citrus. micrornas play important roles in nutrient deficiencies. however, little is known on b-deficiency-responsive mirnas in plants. in this study, we first identified mirnas and their expression pattern in b-deficient citrus sinensis roots by illumina sequencing in order to identify mirnas that might be involved in the tolerance of plants to b-deficiency.  we isolated up-regulated and down-regulated mirnas from b-deficient roots, demonstrating remarkable metabolic flexibility of roots, which might contribute to the tolerance of plants to b-deficiency. a model for the possible roles of mirnas in the tolerance of roots to b-deficiency was proposed. mirnas might regulate the adaptations of roots to b-deficiency through following several aspects: inactivating reactive oxygen species signaling and scavenging through up-regulating mir and down-regulating mir and mir843; increasing lateral root number by lowering mir expression and maintaining a certain phenotype favorable for b-deficiency-tolerance by increasing mir expression; enhancing cell transport by decreasing the transcripts of mir mir and mir3465; improving osmoprotection and regulating other metabolic reactions. other mirnas such as mir and mir in roots increased in response to b-deficiency, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots.  our work demonstrates the possible roles of mirnas and related mechanisms in the response of plant roots to b-deficiency. boron-deficiencyboron-tolerancecitrus sinensisillumina sequencingmicrornareactive oxygen species 