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 (...