Regulation of miR399f Transcription by AtMYB2 Affects Phosphate Starvation Responses in Arabidopsis  

Abstract

Although a role for microRNA399 (miR399) in plant responses to phosphate (Pi) starvation has been indicated, the regulatory mechanism underlying miR399 gene expression is not clear. Here, we report that AtMYB2 functions as a direct transcriptional activator for miR399 in Arabidopsis (Arabidopsis thaliana) Pi starvation signaling. Compared with untransformed control plants, transgenic plants constitutively overexpressing AtMYB2 showed increased miR399f expression and tissue Pi contents under high Pi growth and exhibited elevated expression of a subset of Pi starvation-induced genes. Pi starvation-induced root architectural changes were more exaggerated in AtMYB2-overexpressing transgenic plants compared with the wild type. AtMYB2 directly binds to a MYB-binding site in the miR399f promoter in vitro, as well as in vivo, and stimulates miR399f promoter activity in Arabidopsis protoplasts. Transcription of AtMYB2 itself is induced in response to Pi deficiency, and the tissue expression patterns of miR399f and AtMYB2 are similar. Both genes are expressed mainly in vascular tissues of cotyledons and in roots. Our results suggest that AtMYB2 regulates plant responses to Pi starvation by regulating the expression of the miR399 gene.Phosphorus (P) is an essential component of all organisms, as it is found, among other compounds, in nucleic acids, ATP, and membrane phospholipids. It is an essential nutrient for plants. P can be acquired by plants only as inorganic phosphate (Pi). Therefore, most of the P content of soils is unavailable for plant growth and development (Hinsinger, 2001). To overcome the problem of Pi limitation, plants have developed a variety of adaptive responses that conserve internal P while activating mechanisms that enhance the accessibility and uptake of external P. The accompanying gene expression changes produce changes in root architecture, enhanced Pi uptake activity, secretion of organic acids, and secretion of phosphatases (Raghothama, 1999;Poirier and Bucher, 2002;Yuan and Liu, 2008;Péret et al., 2011). The synchronization of Pi availability with plant growth and development is orchestrated by several phytohormones, including abscisic acid, ethylene, auxin, and cytokinin (Hillwig et al., 2008;Devaiah et al., 2009;Lei et al., 2011).A few transcription factors have been characterized that appear to regulate subsets of the response to Pi stress, either positively or negatively. PHOSPHATE STARVATION RESPONSE1 (PHR1) is a MYB transcription factor that initiates the up-regulation of Pi starvation-responsive genes in plants and unicellular algae (Rubio et al., 2001). WRKY75, a WRKY transcription factor family member, has been identified as a key regulator of Pi acquisition and root architecture in response to Pi starvation (Devaiah et al., 2007a). MYB62, an R2R3-type MYB transcription factor, connects Pi homeostasis and GA signaling during Pi starvation (Devaiah et al., 2009). ZAT6, a C2H2-type zinc finger transcription factor, regulates Pi homeostasis and exerts some control over root development (...

Dynamic roles of microRNAs in nutrient acquisition and plant adaptation under nutrient stress: A review

“…Under P deficiency, Arabidopsis phr1 mutants showed significantly reduced expressions of miR399 as well as group of other P related genes, while set of P deficiency-responsive genes were found upregulated in pho2 mutants under sufficient P availability, hence, concluded that PHR1 functions upstream of miR399 and PHO2, however, it is not the only element required for miR399 induction in P signaling network (Bari et al, 2006). Another recent report conducted by Baek et al (2013), demonstrated that miR399f belongs to Arabidopsis, transcriptionally regulated by MYB TF induced in P-deprived plants thereby binding to a MYB binding-site present in the promoter region of miR399f. Previous compelling evidences demonstrated that miR399 act as positive regulator under P deficiency, while PHO2; a target gene for miR399, behave as negative regulator and inhibit P uptake under normal P concentrations (Lin et al, 2008;Yuan and Liu, 2008).…”

Insights into the Small RNA-Mediated Networks in Response to Abiotic Stress in Plants

“…All miR399 genes present in Arabidopsis harbor PHR1-binding site P1BS in their promoters (Bari et al 2006 ). Also several other P-responsive cis -regulatory elements were identifi ed in the promoters of these phosphate-responsive miRNAs including PHO, PHO-like, W-box, NIT2, and MYB-binding sites (Zeng et al 2010 ;Xu et al 2013 ;Baek et al 2013 ). Further fi ndings also indicated that miR399-mediated regulation of PHO2 is under the control of target mimicry.…”

“…Several abiotic stress-associated transcription factorbinding motifs are identifi ed in the regulatory region of stress-responsive miRNAs in rice (Devi et al 2013 ) and in Arabidopsis (Megraw et al 2006 ). Several individual miRNA studies also identifi ed the cis -acting elements in their upstream region, e.g., ABA-mediated response of AtMIR168a is due to the presence of two ABRE in upstream region (Li et al 2012a ), the presence of PHR1-binding site and other phosphate-responsive cis regulatory elements in the promoter of phosphateresponsive miRNAs (Bari et al 2006, Zeng et al 2010, Baek et al 2013 ) and core GTAC motif as AtSPL7-binding site in the upstream regulatory region of copper-responsive miRNAs (Yamasaki et al 2009 ). Also the LONG HYPOCOTYL 5 (AtHY5) acting downstream of many families of photoreceptors, binds to the upstream region of several miRNAs (miR156d, miR172b, miR402, miR408, miR775, miR858, miR869, and miR1888) (Zhang et al 2011a ).…”

“…Other phosphate limitation-responsive miRNAs, miR169, miR827, and miR2111 were also detected in the phloem sap of rape seed suggesting that the systemic movement of these small RNAs function in the regulation of phosphate homeostasis (Pant et al 2009 ). Transcriptional activation of miR399 was regulated by MYB transcription factors, PHR1 (Phosphate starvation response 1), PHL1 (PHR1 like 1), and MYB2 (Bustos et al 2010 ;Rubio et al 2001 ;Baek et al 2013 ). All miR399 genes present in Arabidopsis harbor PHR1-binding site P1BS in their promoters (Bari et al 2006 ).…”

Phosphate starvation induces DNA methylation in the vicinity of cis-acting elements known to regulate the expression of phosphate–responsive genes

“…A number of transcription factors including the master regulator PHOSPHATE RESPONSE 1 (PHR1), and several members of the MYB and WRKY families of transcription factors have been shown to regulate the transcriptional activation of lowphosphate responsive genes and their corresponding binding sites P1BS, MBS and the W-box, respectively, act as cis-acting elements that mediate Pi starvation-responsive expression. [15][16][17][18][19][20] A search for these cis-acting motifs in Pi-methDEGs showed that 39 of these genes contained P1BS motifs, that present the palindromic 8 base pair sequence (GNATATNC) where PHR1 binds to DNA as a dimer, 15 93 genes had W-box elements which are the cognate binding sites of WRKY transcription factors, which have been characterized as key regulators of several processes in plants, including some members of this family which are specifically involved in the response to low Pi conditions, 17,19,21,22 and 80 genes contained MYB transcription factors Binding sites (MBS) (Fig. 1B).…”