The Role of MicroRNAs in Phosphorus Deficiency Signaling

As an essential macronutrient for plant growth, development, and reproduction, phosphorus (P) is a basic component of cellular structures, functions in energy metabolism and signal transduction cascades, and regulates enzymatic activities. Despite the abundance of P in the soil, the form of P available for uptake by plants, orthophosphate (Pi), is usually present at a low level in the soil due to its precipitation with cations and conversion to organic matter (Marschner, 1995). To circumvent the limited availability of Pi, plants have evolved a repertoire of adaptive responses, so-called P starvation responses, which involve diverse developmental and biochemical processes, to increase Pi acquisition and utilization (Raghothama, 1999). To initiate these adaptive responses, P deficiency signaling is transduced via multiple regulatory routes, of which transcriptional or posttranscriptional regulation of gene expression and posttranslational regulation of protein activities have been documented (Franco-Zorrilla et al., 2004; Miura et al., 2005; Chiou and Lin, 2011). While several key transcription factors responsible for the transcriptional control of P starvation-responsive (PSR) genes have been identified, microRNAs (miRNAs) have emerged as new players in regulating PSR genes at the posttranscriptional level. miRNAs are endogenous noncoding RNAs 20 to 24 nucleotides in size that are generated from a single-stranded RNA precursor with a hairpin secondary structure. miRNAs negatively regulate gene expression at the posttranscriptional level by base-pairing to their target mRNAs, which often directs mRNA cleavage in plants (Reinhart et al., 2002). Over the past few years, collective studies have revealed significant roles of miRNAs in P deficiency signaling and regulation of Pi homeostasis through the identification and characterization of PSR miRNAs. In this update, we will focus on the roles of PSR miRNAs as effectors and transmitting molecules in P deficiency signaling, the implications of diverse PSR miRNAs in P starvation adaptive responses, and their regulation in response to P deficiency.

ROLES OF MIR399 IN REGULATING PHOSPHATE HOMEOSTASIS

miR399 is the first miRNA demonstrated to be up-regulated specifically by P deficiency and rapidly decreased upon P readdition (Fujii et al., 2005). The Arabidopsis (Arabidopsis thaliana) genome encodes six MIR399 genes (MIR399A to -F), which are all induced by P starvation to different extents. Transgenic Arabidopsis plants overexpressing miR399 exhibit increased Pi uptake and allocation to the shoot, resulting in excessive shoot Pi when grown under P-sufficient conditions (Fujii et al., 2005; Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006). The high Pi level is accompanied by chlorosis and necrosis at the tips of mature leaves, which are characteristics of Pi toxicity (Delhaize and Randall, 1995). Remobilization of Pi from old to young leaves during P starvation is impaired in the transgenic lines (Chiou et al., 2006). Similar phenotypes of Pi toxicity and defective Pi allocation are observed in rice (Oryza sativa) transgenic plants overexpressing the miR399 homologs (Hu et al., 2011). Overexpression of Arabidopsis miR399 in tomato (Solanum lycopersicum) not only results in increased accumulation of Pi but also enhances the secretion of acid phosphatase and proton in the roots, which facilitates the hydrolysis of soil organic P and dissolution of Pi (Gao et al., 2010). Therefore, miR399 plays important roles in maintaining Pi homeostasis at the level of Pi acquisition, allocation, and remobilization. Upon P deficiency, miR399 acts as a positive regulator to promote Pi uptake and root-to-shoot allocation of Pi.

In Arabidopsis, three genes are predicted to be targets of miR399: they encode a Pi transporter (PHT1;7), a DEAD box helicase, and a ubiquitin-conjugating E2 enzyme; however, only the E2 enzyme encoded by UBC24 has been experimentally validated (Allen et al., 2005). UBC24 is expressed abundantly in the root under P-sufficient conditions and is down-regulated in response to P starvation, which coincides temporally with the up-regulation of miR399s (Fujii et al., 2005; Bari et al., 2006; Chiou et al., 2006). Multiple sequences complementary to miR399 in the 5′ untranslated region (UTR) of UBC24 direct the cleavage of UBC24 by miR399. The level of UBC24 mRNA is largely reduced in transgenic plants overexpressing miR399, whereas accumulation of UBC24 mRNA persists in those plants that overexpress mutated miR399 (Fujii et al., 2005). Consistent with these results, mRNAs of the UBC24 transgene with deletions of miR399 complementary sequences at the 5′ UTR are stable under P starvation (Fujii et al., 2005).

In accordance with being antagonized by miR399s, UBC24 negatively regulates Pi uptake and root-to-shoot allocation. The Arabidopsis T-DNA knockout ubc24 mutant exhibits the same phenotypes observed in the miR399-overexpressing transgenic plants (Chiou et al., 2006). Phenotypes of the miR399-overexpressing transgenic Arabidopsis plants and ubc24 mutants resemble those of a previously reported Pi overaccumulator, the pho2 mutant (Delhaize and Randall, 1995; Dong et al., 1998). Subsequent characterization confirmed that pho2 is caused by a nonsense mutation in the UBC24 gene (Aung et al., 2006; Bari et al., 2006). Micrografting experiments between wild-type Arabidopsis plants and pho2 mutants showed that the loss-of-function mutation of PHO2 in the roots is sufficient to cause Pi overaccumulation in the shoots, whereas a pho2 mutation in the scion does not cause such a phenotype (Bari et al., 2006; Lin et al., 2008). These observations indicate that PHO2/UBC24 functions mainly in the roots to regulate Pi acquisition and root-to-shoot Pi allocation.

The molecular mechanism by which PHO2/UBC24 negatively regulates Pi uptake and allocation remains elusive. Among the genes encoding plasma membrane-localized Pi transporters that were examined in Arabidopsis, the transcripts of PHT1;8 and PHT1;9 are significantly increased in the pho2 mutant roots (Aung et al., 2006; Bari et al., 2006). Thus, PHT1;8 and PHT1;9 are suspected to function downstream of PHO2 and may be responsible for the overaccumulation of Pi in the pho2 mutant. However, whereas the RNA interference knockdown of PHT1;8 suppresses Pi accumulation in the pho2 mutant, T-DNA knockout mutations of PHT1;8 and/or PHT1;9 in the pho2 mutant do not affect Pi accumulation (Bari et al., 2006; Y.-S. Lai, P.-C. Wu, and T.-J. Chiou, unpublished data). The reasons for this discrepancy are unclear, and the roles of PHT1;8 and PHT1;9 in Pi accumulation in the pho2 mutant require further assessment. Other than plasma membrane-targeted Pi transporters, several intracellular Pi transporter-coding genes, such as PHT2;1, PHT3;1, and PHT3;2, are up-regulated in the shoots of pho2 mutants, which may reflect a cellular adjustment resulting from elevated intracellular Pi concentrations (Aung et al., 2006). Increased expression of genes coding for several Pi transporters, a phosphatase, and ribonucleases is also found in the roots of rice pho2 mutants, further demonstrating the importance of PHO2/UBC24 in the regulation Pi homeostasis (Liu et al., 2010a; Hu et al., 2011). As a ubiquitin-conjugating E2 enzyme, it is likely that PHO2/UBC24 modulates protein activities through degradation, and the transcriptional alteration observed in the pho2 mutant could be explained as a secondary or indirect effect. It is worth noting that the enhanced Pi uptake activity in the pho2 mutant is a result of increased V_max without changes in K_m, suggesting that the amount of transport proteins is increased (Aung et al., 2006). Identification of the target proteins subject to PHO2/UBC24 modifications is essential to delineate the signaling pathway mediated by miR399 and PHO2/UBC24.

Homologs of miR399s have been identified in diverse angiosperm species, such as rice, tomato, common bean (Phaseolus vulgaris), and Medicago truncatula (Table I). The responses of miR399 homologs to P deficiency have also been experimentally validated in several species, indicating that miR399 functions as a conserved riboregulator of P deficiency signaling. Accordingly, genes homologous to PHO2/UBC24 have been found in corresponding species and have a conserved structure, with multiple miR399-complementary sequences residing in the 5′ UTR (Bari et al., 2006; Lin et al., 2008). The inverse relationship of expression patterns between miR399 and UBC24 homologs in response to Pi starvation has also been observed in rice and common bean (Valdés-López et al., 2008; Liu et al., 2010b; Hu et al., 2011). Therefore, the miR399-PHO2 regulatory mechanism in Pi homeostasis is evolutionarily conserved in angiosperms.

Table I. Summary of validated PSR miRNA families.

The expression of these listed miRNA families has been validated by qRT-PCR or northern-blot analysis following preliminary screens by deep sequencing, microarray, or macroarray analysis.

miRNA Familya	Target Gene Productb	Speciesc	Expression in Response to Pi Deficiencyd	Referencee
156	SPL transcription factors	At	R(+)	6
		La	R(+), L(−)	16
157	SPL transcription factors	Phv	N(+)	13
158	–	Sl	R(−), L(−)	5
159	MYB, TCP transcription factors	La	R(+), SM(−), L(−)	16
		Gm	R(+)	14
160	Auxin response factors (ARFs)	La	R(+), L(−)	16
163	–	At	S(+)	9
164	NAC transcription factors	La	R(+), S(−), L(−)	16
166	HD-ZIP transcription factors	La	R(+), SM(−), L(−)	16
		Gm	R(−)	14
167	Auxin response factors (ARFs)	La	R(+), L(−)	16
168	Argonaute (AGO1)	La	R(+), L(+)	16
169	HAP2 transcription factors	At	SD(−), R(−), S(−)	6, 9, 11
169g*	–	Sl	L(−)	5
171	SCARCROW-like transcription factors	La	SM(+), L(+)	16
172	AP2 transcription factors	Sl	L(−)	5
172b*	–	Sl	L(−)	5
319	TCP transcription factors	La	R(+), SM(−)	16
		Sl	R(+), L(−)	5
		Gm	R(−)	14
390	Auxin response factors (ARFs)	La	R(−)	16
394	F-box proteins	Sl	R(+)	5
395	ATP sulfurylase; sulfate transporter	At	R(−), S(−)	6
		La	R(−), SM(+), L(+)	16
396	Growth-regulating factor (GRF)	La	R(+), L(−)	16
397	Laccases; β-6 tubulin	La, Phv	L(−)	16, 13
398	Copper/zinc superoxide dismutase;	At	SD(−), R(−), S(−)	6, 11
	cytochrome c oxidase subunit V	Sl	L(+)	5
		Gm	R(−)	14
		Phv	L(−)	13
399	Ubiquitin conjugase E2/UBC24	At	SD(+), R(+), S(+)	1, 4, 6, 9, 11, 14
		Mt, Phv	R(+), L(+)	2, 8, 12
		Pav	SDL(+)	10
		La	L(+)	16
		Sl	R(+), SM(+), L(+)	4, 5
		Os	R(+), S(+)	1, 4, 15
399*	–	At	SD(+), R(+), S(+)	6, 11
402	–	At	R(−)	6
437	–	La	R(+)	16
447	–	At	SD(+)	11
		La	SM(+), L(+)	16
472	–	La	SM(+)	16
477	–	La	L(+)	16
530	–	La	L(−)	16
771	–	Sl	L(−)	5
775	–	Sl	L(−)	5
778	SET domain-containing protein	At	SD(+),R(+), S(+)	6, 11
778*	–	At	SD(+)	11
818	–	La	R(−), SM(+), L(+)	16
827	Ubiquitin E3 ligase NLA	At	SD(+), R(+), S(+)	6, 7, 9, 11
	SPX-MFS1, SXP-MFS2	Os	R(+), S(+)	7
828	TAS4	At	S(+)	6
830	–	La	R(+)	16
837	–	Sl	L(−)	5
845	–	La	R(+)	16
862	–	Sl	R(−)	5
866	–	La	SM(+), L(+)	16
893	–	La	R(−)	16
895	–	La	R(+), SM(−)	16
896	–	La	R(+), L(−)	16
903	–	La	SM(+), L(+)	16
904	–	La	SM(+)	16
1211	–	La	R(−)	16
1222	–	La	R(+)	16
2111-5p	F-box protein	At	SD(+), R(+), S(+)	6, 11
2111-3p	–	At	SD(+), R(+), S(+)	6, 11

^amiRNA families identified in multiple species (including those listed in Table II) are shown in boldface.

^bListed are experimentally validated genes as determined by 5′ RACE analysis. Dashes denote that no target genes have been validated.

^cAt, Arabidopsis; Bn, rapeseed; Gm, soybean; La, white lupin; Mt, M. truncatula; Os, rice; Pav, switchgrass (Panicum virgatum); Phv, common bean; Sl, tomato.

^d+ denotes up-regulated expression and − denotes down-regulated expression. L, Leaf; N, nodule; PS, phloem sap; R, root; S, shoot; SD, seedling; SM, stem.

^eCorresponding references are as follows: 1, Bari et al. (2006); 2, Branscheid et al. (2010); 3, Buhtz et al. (2008); 4, Chiou et al. (2006); 5, Gu et al. (2010); 6, Hsieh et al. (2009); 7, Lin et al. (2010); 8, Liu et al. (2010b); 9, Lundmark et al. (2010); 10, Matts et al. (2010); 11, Pant et al. (2009); 12, Valdés-López et al. (2008); 13, Valdés-López et al. (2010); 14, Zeng et al. (2010); 15, Zhou et al. (2008); 16, Zhu et al. (2010).

DIVERSE FAMILIES OF MIRNAS REGULATED BY P DEFICIENCY

Following the identification of miR399, an expanding list of PSR miRNA families has been identified in several plant species by various approaches, including microarrays, high-throughput small RNA sequencing, quantitative reverse transcription (qRT)-PCR, and northern-blot analyses (Tables I and II; see refs. cited therein). Table I summarizes the PSR miRNAs whose expression has been experimentally validated either by qRT-PCR or northern-blot analysis, while Table II lists the PSR miRNA candidates identified from the preliminary analysis of deep sequencing, microarray, or macroarray. As summarized in the tables, P deficiency triggers expression changes in diverse families of miRNAs. It is notable that certain miRNA families are commonly responsive to P deficiency among species, such as miR156, miR159, miR166, miR319, miR395, miR398, miR399, miR447, and miR827. These miRNAs are presumably involved in conserved P deficiency signaling pathways in plants, as exemplified by miR399. On the other hand, the responsiveness of many miRNAs appears to be species specific; these miRNAs are likely recently evolved and their functions in P deficiency adaptation still need to be evaluated. We should also note that miRNAs may respond differently in different tissues/organs to P deficiency and that such tissue-specific responsiveness could vary among species. For example, miR395 is down-regulated in the shoots of Arabidopsis but up-regulated in the shoots of white lupin (Lupinus albus) upon P starvation (Table I). How signals of P deficiency are sensed in different tissues/organs and transduced into different transcriptional responses, as well as the outcome of such coordination of expression between tissues/organs, remain to be investigated. The majority of validated PSR miRNA target genes are involved in transcriptional regulation, such as transcription factors (Table I). Others include genes coding for biotic/abiotic stress-responsive proteins and enzymes involved in protein modification/degradation. The diverse functions of these targets may be necessary to coordinate P deficiency responses by orchestrating a broad range of biological processes.

Table II. List of candidate PSR miRNA families.

These miRNA families were identified by preliminary analysis (e.g., deep sequencing, microarray, or macroarray analysis) but have not been further validated.

miRNA Familiesa	Species	Referenceb
157, 159, 160, 164, 165, 166, 167, 168,171, 172, 319, 391, 393, 398*, 408, 408*, 779, 780, 822, 823, 824, 829, 843, 860, 863, 865, 866	At	6, 9, 11
169, 399, 399*, 827, 2111-5p, 2111-3p	Bn	3, 11
156, 157, 160, 165, 167, 168, 396, 474, 482, 834, 845, 854, 894, 1118, 1311, 1427, 1436, 1450, 1507, 1508, 1509, 1511, 1846, 1858, 1879, 1881	Gm	14
156, 159, 160, 165, 166, 167, 169, 170,172, 319, 393, 408, 1508, 1509, 1511, 1513, 1514, 1515, 1516, 1524, 1526, 1532, 2118, 2119	Phv	13

^amiRNA families identified in multiple species are shown in boldface.

^bNumbers of references correspond to those given in Table I.

Like miR399, the two miRNAs miR827 and miR2111 are highly and specifically induced by P deficiency but not by other nutrient deficiencies in Arabidopsis (Hsieh et al., 2009). The target genes of miR827 encode proteins containing the SPX domain. SPX domain proteins have been implicated in Pi transport or sensing in yeast and in responding to changes in external P concentration or xylem loading of Pi in plants (Lenburg and O’Shea, 1996; Hamburger et al., 2002; Duan et al., 2008). Arabidopsis miR827 (ath-miR827) targets the NITROGEN LIMITATION ADAPTATION (NLA; also named BENZOIC ACID HYPERSENSITIVE1) transcript coding for a protein consisting of an N-terminal SPX domain and a C-terminal RING domain, the latter of which possesses a ubiquitin E3 ligase activity (Peng et al., 2007; Yaeno and Iba, 2008; Hsieh et al., 2009; Pant et al., 2009). The expression of NLA is down-regulated under Pi deficiency, reciprocal to that of ath-miR827 (Hsieh et al., 2009; Kant et al., 2011). NLA was first described to be involved in the adaptive responses to nitrogen (N) deficiency, and nla mutants exhibit earlier senescence than wild-type plants under N limitation (Peng et al., 2007). However, a very recent report showed that mutations in PHOSPHATE TRANSPORTER FACILITATOR1 (PHF1) or PHT1;1 Pi transporter suppress the early-senescence phenotype of nla mutants in response to N deficiency (Kant et al., 2011). Further analyses indicated that such an early-senescence phenotype of the nla mutant is in fact caused by excessive Pi accumulation, pointing out the importance of NLA in maintaining Pi homeostasis in a nitrate-dependent manner (Kant et al., 2011). Consistent with the negative regulation of NLA by miR827, miR827-overexpressing transgenic plants, resembling nla mutants, accumulate high levels of Pi under low-N conditions. It is worth noting that pho2 mutants also exhibit a similar nitrate-dependent Pi overaccumulation phenotype; moreover, nla pho2 double mutants show a comparable extent of Pi accumulation as nla and pho2 mutants, suggesting that NLA and PHO2 may function in the same pathway (Kant et al., 2011). It has been hypothesized that PHO2 and NLA, under the respective regulation by miR399 and miR827, coordinate to suppress Pi uptake by the degradation of proteins such as PHF1 and Pi transporters through the ubiquitination pathway.

A more complex regulation of miR827 and its target genes in rice was also reported. Similar to Arabidopsis, the rice miR827 (osa-miR827) is also up-regulated by P deficiency, but its target transcripts are different from those in Arabidopsis. osa-miR827 cleaves OsSPX-MFS1 and OsSPX-MFS2 mRNAs, which encode proteins with an N-terminal SPX domain and a C-terminal MFS domain involved in solute transport (Marger and Saier, 1993; Lin et al., 2010). Studies on transgenic rice overexpressing or abolishing osa-miR827 expression demonstrated that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827. Interestingly, OsSPX-MFS1 is down-regulated, whereas OsSPX-MFS2 is up-regulated, in response to P deficiency. Because osa-miR827 is coexpressed with both OsSPX-MFS1 and OsSPX-MFS2 in the same cell types, it may play roles in suppressing or fine-tuning the expression of these target genes during P deficiency (Lin et al., 2010).

Unlike most miRNAs, for which one strand of the small RNA duplex (i.e. miRNA*) is rapidly degraded, both complementary strands of the miR2111 small RNA duplex accumulate to substantial amounts (Hsieh et al., 2009; Pant et al., 2009). Among several predicted transcript targets of miR2111, cleavage of a transcript coding for an F-box protein, a component of SCF ubiquitin ligase complexes, has been experimentally validated; however, its transcript level does not show a negative correlation with miR2111 during P starvation (Hsieh et al., 2009). It is worth noting that the target genes of three highly inducible miRNAs under P deficiency (i.e. miR399, miR827, and miR2111) all encode proteins involved in the ubiquitin-mediated protein degradation pathway, indicating the importance of posttranslational regulation of protein levels in the adaptive responses of P deficiency.

PSR miRNAs also regulate noncoding transcripts. Induced by Pi deficiency, miR828 not only targets the transcript of a MYB transcription factor (MYB113) but also the TAS4 transcript that produces clusters of phased transacting, small interfering RNAs (ta-siRNAs; Rajagopalan et al., 2006; Hsieh et al., 2009). TAS4-siR81(−), one of the dominant TAS4 siRNAs, targets the transcripts of a group of MYB transcription factors, PAP1/MYB75, PAP2/MYB90, and MYB113, which are involved in anthocyanin biosynthesis (Rajagopalan et al., 2006). Because anthocyanin accumulates during P starvation, the positive correlation between the expression of miR828/TAS4-siR81(−) and their target genes suggests an autoregulatory mechanism that modulates anthocyanin accumulation. Under this mechanism, P deficiency activates the expression of PAP1, PAP2, and MYB113, resulting in the biosynthesis of anthocyanin. PAP1 subsequently activates miR828 (and likely TAS4), leading to the production of TAS4-siR81(−), which in turn suppresses the transcript level of the MYB genes (Hsieh et al., 2009). Because anthocyanin accumulation is a common stress response and TAS4-siR81(−) is also induced under N deficiency, such an autoregulation mechanism may apply to other stress conditions (Hsieh et al., 2009).

The observation that three dominant P deficiency-down-regulated miRNAs, miR169, miR395, and miR398, are also responsive to other nutritional deficiencies (e.g. N, potassium, copper, iron, or sulfur deficiency) suggests that miRNAs are involved in stress signal transduction pathways that cross talk with different nutrient homeostasis. The down-regulation of miR395 during Pi starvation is associated with an up-regulation of its target genes, APS4 and SULTR2;1, which may result in an increase in sulfate assimilation and translocation that in turn facilitates sulfolipid biosynthesis to replace reduced phospholipids during Pi deficiency (Hsieh et al., 2009). Down-regulation of miR169 and miR398 is likely involved in the relief of oxidative stress accompanied by nutrient deficiency, as the target gene product of miR169, the transcription factor HAP2, activates several drought-responsive genes, and the miR398 target gene encodes a copper/zinc superoxide dismutase that removes reactive superoxides (Sunkar et al., 2006; Li et al., 2008).

SYSTEMIC ROLES OF MIRNAS IN RESPONSE TO P DEFICIENCY

Coordinated regulation of Pi uptake in the roots and of Pi allocation between organs is essential for proper plant growth and development, particularly in response to fluctuating external P supply. Studies of miR399 and PHO2 in Arabidopsis, rapeseed (Brassica napus), pumpkin (Cucurbita maxima), and tobacco (Nicotiana tabacum) indicate that miR399 acts as a mobile signaling molecule in coordinating P homeostasis between the aerial tissues and the underground roots. First, miR399 accumulates in the phloem sap and to a higher abundance in response to P deficiency (Buhtz et al., 2008; Pant et al., 2008). Second, miR399 genes are expressed in the vascular tissues (Aung et al., 2006). Strong evidence for the shoot-to-root movement of miR399 has been provided by micrografting experiments, in which mature miR399 was detected in the wild-type rootstocks grown under P-sufficient conditions when grafted with miR399 overexpression scions (Lin et al., 2008; Pant et al., 2008). The accumulation of mature miR399 does not result from de novo transcription in the rootstocks because primary transcripts of miR399 (pri-miR399s) are not detectable. Like miR399-overexpressing transgenic plants and pho2 mutants, accumulation of miR399 in the wild-type rootstocks is accompanied by decreased PHO2 mRNA and increased Pi levels in the miR399-overexpressing scions (Lin et al., 2008; Pant et al., 2008).

The movement of miR399 occurs from the shoot to the root but not vice versa, because primary transcripts and mature miR399 remained undetectable in the wild-type shoot scions grafted to the miR399-overexpressing rootstocks (Lin et al., 2008; Pant et al., 2008). Therefore, miR399 does not translocate through the xylem; this observation is consistent with the fact that xylem sap is devoid of small RNAs (Buhtz et al., 2008). Compared with the roots, plants appear to respond to Pi limitation more readily in the shoots, where pri-miR399s are up-regulated first, while the mature miR399s accumulate at higher levels in the roots (Bari et al., 2006; Lin et al., 2008). It has been hypothesized that upon Pi starvation, shoot-induced miR399s are translocated via the phloem to target PHO2 mRNA in the roots, which in turn regulates Pi uptake and allocation to the shoots, and such long-distance communication is a crucial early response to P deficiency. Taken together, these results indicate that miR399 functions as a systemic signaling molecule that involves the coordination of whole-plant Pi homeostasis. Although several miR399 species (including miR399b, -c, -d, and -f) are able to translocate to the grafted roots when overexpressed in the shoots, it is unclear whether there is any preference of specific miR399 species for such systemic translocation in the wild-type plants (Lin et al., 2008; Pant et al., 2008). In addition to miR399, the miRNAs miR169, miR827, and miR2111 were detected to be responsive to Pi limitation in the phloem sap of rapeseed, suggesting that multiple PSR miRNAs are involved in systemic signaling to regulate various adaptive processes during Pi starvation (Pant et al., 2009). It is likely that miRNA-mediated systemic signaling is a general mechanism underlying nutrient deficiency responses, as miR395 and miR398 also accumulate in the phloem sap of rapeseed in response to sulfur and copper deprivation, respectively (Buhtz et al., 2008, 2010).

Interestingly, miRNA* strands of certain PSR miRNA species have been detected in Arabidopsis tissues and in the phloem sap of rapeseed (Tables I and II; Buhtz et al., 2008; Hsieh et al., 2009; Pant et al., 2009). Because several of these miRNA* species (e.g. miR399*, miR398*, and complementary strands of miR2111) are present in significant abundance and are responsive to the Pi status, it is likely that they are of biological relevance (Buhtz et al., 2008). Moreover, like miR399, miR399* can also move across the grafting junction (Lin et al., 2008). It is speculated that miRNA* strands are involved in systemic signaling by forming duplexes with their complementary miRNAs for movement, as in the case of cell-to-cell movement of siRNA duplexes in gene silencing (Dunoyer et al., 2010). Although an in vitro RNase digestion assay on isolated phloem sap RNAs showed that these miRNA* strands are predominantly single stranded, the possibility that miRNA duplexes dissociated during RNA preparation needs to be carefully assessed (Buhtz et al., 2008). Because no target genes of these miRNA* strands have been validated so far, whether these miRNA* strands play any role in gene regulation requires further investigation.

REGULATION OF P STARVATION-RESPONSIVE MIRNAS

The significant roles of miRNAs in mediating systemic signaling as well as in regulating genes involved in a broad range of biological functions during P deficiency suggest a delicate control of these miRNAs in a spatial and/or temporal manner. The MYB transcription factors PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHR1-Like1 (PHL1) are central integrators of P deficiency signaling in transcriptional activation of a broad range of PSR genes (Bustos et al., 2010). The PHR1-binding site P1BS is present in the promoters of all six miR399 genes as well as other PSR miRNA genes, such as miR778 and miR827 (Bari et al., 2006; Hsieh et al., 2009). Expression of miR399 primary transcripts is reduced in the phr1 mutant, suggesting that miR399 is regulated by PHR1 in addition to other regulatory factors (Bari et al., 2006). The expression of miR399 primary transcripts is also positively regulated by the overexpression of the rice PHR1 homolog, OsPHR2 (Zhou et al., 2008). To investigate the transcriptional regulation of PSR miRNAs, a computational analysis of Pi-responsive cis-elements in promoter sequences was conducted in soybean (Glycine max; Zeng et al., 2010). The analysis revealed an overrepresentation of P1BS, PHO, W-box, and NIT2 elements. Although further experimental support will be required, the presence of diverse cis-elements in the promoter region suggests multiple and coordinated actions of various transcription factors in regulating the transcription of PSR miRNAs.

Photosynthetic carbon assimilation has been shown to cross-regulate P deficiency signaling (Liu et al., 2005; Karthikeyan et al., 2007). Photosynthates or sugars are required for the induction of miR399 expression under P deficiency (Liu et al., 2010b). Expression of miR399 is suppressed in the leaves of P-starved common bean plants subjected to dark treatment but not in the leaves exposed to light. The effects of photosynthates are systemic, as continuous darkness or stem-girdling treatment of common bean plants fails to induce miR399 expression in the roots upon P starvation. Although it remains unclear how photosynthates cross-regulate the signaling of P starvation, current observations indicate that they act upstream of the P deficiency signaling pathway.

The establishment of symbiosis with arbuscular mycorrhizal (AM) fungi is a common adaptive P starvation response, in which plants increase P acquisition assisted by the symbiotic fungi (Javot et al., 2007; Parniske, 2008). It is of interest to know whether AM symbiosis can regulate the expression of PSR miRNAs. In M. truncatula, the accumulation of most miR399 primary transcripts in roots is reduced in response to AM symbiosis, likely due to locally increased Pi. On the contrary, mature miR399s accumulate to a high level in the mycorrhizal roots (Branscheid et al., 2010). Because the primary transcripts of five miR399 genes are up-regulated in the leaves of mycorrhizal plants, it is postulated that a mycorrhiza-induced signal derived from the roots may activate miR399 transcription in the shoots and that the processed mature miR399s are subsequently translocated to the roots to suppress PHO2 expression (Branscheid et al., 2010). The nature of such symbiotic signals that regulate miR399 expression and the roles of PHO2 in symbiotic P acquisition remain to be identified.

Studies of a noncoding RNA family have revealed an alternative regulation mechanism of miR399. A group of noncoding RNAs (e.g. TPSI1 in tomato, IPS1 and At4 in Arabidopsis, and Mt4 in M. truncatula) ranging from 500 to 700 nucleotides in length are highly induced by P starvation (Burleigh and Harrison, 1997; Liu et al., 1997; Martín et al., 2000; Shin et al., 2006). These noncoding RNAs share very limited sequence similarity, with the exception of a 22- to 24-nucleotide-long sequence motif that is partially complementary to miR399 (Shin et al., 2006; Franco-Zorrilla et al., 2007). The IPS1/At4 members are not the targets of miR399, but their overexpression attenuates the suppressive effect of miR399 on PHO2 mRNA and reduces the Pi accumulation in miR399-overexpressing transgenic plants, thus suggesting that IPS1/At4 counteracts miR399 activity. Consistent with these observations, loss-of-function mutations in At4 result in increased shoot Pi content upon P limitation (Shin et al., 2006; Franco-Zorrilla et al., 2007).

The mechanism by which IPS1/At4 inhibits miR399 activity is based on a direct interaction between RNA molecules. Mutations that result in perfect base-pairing between IPS1 and miR399 render the IPS1 transcript susceptible to cleavage by miR399, and the inhibitory effect of IPS1 on miR399 activity is lost (Franco-Zorrilla et al., 2007). Therefore, it has been proposed that the IPS1/At4 family members function as target mimics that modulate miR399 activities by sequestering miR399 from binding to PHO2 mRNA and fine-tune the regulation of downstream P starvation responses and Pi homeostasis. This model is supported by the finding that At4 and miR399 coexpress in the root vascular tissues during Pi starvation (Shin et al., 2006). In addition, a moderate level of PHO2 transcript is retained (likely for executing other biological functions) in the tissues of wild-type plants under Pi starvation, despite the presence of an abundant level of miR399 (Aung et al., 2006; Bari et al., 2006). Target mimicry-based regulation of miR399 could be a conserved mechanism, since homologs of the IPS1/At4 genes are present in many plant species. Although the prevalence of natural miRNA target mimics in plants is uncertain, artificial miRNA target mimics have been designed and employed to suppress the activity of several miRNA families for functional studies (Todesco et al., 2010).

Results from grafting experiments indicate that miR399b/c are less efficient in cleaving PHO2 mRNA than miR399f, which is likely due to a difference in nucleotide 13 between miR399s (Lin et al., 2008). Notably, such sequence discrepancy results in reduced base-pairing of miR399b/c to PHO2 mRNA but increased base-pairing to the mimicry sequence of IPS1/At4 (and vice versa in the case of miR399f). Whether this phenomenon has any relevance in gene regulation is not clear.

OTHER P STARVATION-RESPONSIVE SMALL RNAS

In addition to miRNAs, other classes of small RNAs have been shown to respond to P deficiency, such as small RNAs derived from transposons and tRNA-derived small RNAs, suggesting a greater repertoire of small RNA-mediated regulation underlying the P deficiency response (Hsieh et al., 2009). In Arabidopsis, a class of small RNAs with unknown functions, named smRPi1^LTR, that are highly induced under P deficiency, are generated from an intergenic region containing an array of truncated Copia95 retrotransposon-derived long terminal repeats (LTRs). tRNA-derived small RNAs have been identified in various organisms from bacteria to humans (for review, see Hsieh et al., 2010). The class of tRNA-derived small RNAs (19 nucleotides in length) identified in Arabidopsis corresponds to the 5′ end of many tRNA species, and a similar class of tRNA-derived small RNAs was also identified in human cells (Cole et al., 2009; Lee et al., 2009). These small RNAs are present abundantly in the roots under P-replete conditions, and their level is further increased upon P starvation. Small RNAs of tRNA halves generated from the cleavage at the anticodon loop region were also found in the phloem sap of pumpkin and shown to possess an inhibitory activity on protein translation in vitro (Zhang et al., 2009). In animals, tRNA-derived RNA fragments have been implicated in the regulation of cellular proliferation and/or the homeostasis of RNA silencing (Lee et al., 2009; Haussecker et al., 2010). The biological function of plant tRNA-derived small RNAs awaits exploration.

CONCLUSIONS AND PERSPECTIVES

The roles of miRNA in various physiological processes have drawn considerable attention since their discovery, and rigorous research on this topic is ongoing. Current studies have indicated that miRNAs not only function as riboregulators that regulate many downstream genes involved in a broad range of biological processes but also as signal-transmitting molecules that coordinate systemic adaptive responses to sustain proper growth and development of plants under unfavorable conditions. With the aid of high-throughput sequencing technology and computational analysis tools, PSR miRNAs have been identified on a genome-wide scale and their target genes have been predicted. Although information regarding the roles of most PSR miRNAs is currently limited, functional characterization of their target genes will surely provide profound insights into the regulatory mechanisms underlying the adaptive responses to P deficiency.

P is a limiting macronutrient for plant growth and hence affects crop quality and production. The overuse of fertilizer results in severe environmental pollution and reduced biodiversity, whereas in developing countries, agronomic productivity is often restricted by barren farmland and limited access to fertilizer. These problems can be tackled by improving the management of P fertilization and by generating crops with greater efficiency in P uptake and utilization. Current studies indicate that miRNAs play pivotal roles in controlling the adaptive responses to P deficiency. Thus, miRNA-based engineering or miRNA-implemented molecular breeding holds potential to improve crop yields with less application of fertilizer in the future.