Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress.

Summary: Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro‐elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX‐EXS, SPX‐MFS and SPX‐RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

Summary: Arabidopsis thaliana SPX (SYG/PHO81/XPR1) domain genes have recently been shown to be involved in the phosphate (Pi) signaling pathway. We show here that a rice (Oryza sativa) SPX gene, OsSPX1, is specifically induced by Pi starvation in roots. Suppression of OsSPX1 by RNA interference resulted in severe signs of toxicity caused by the over‐accumulation of Pi, similar to that found in OsPHR2 (phosphate starvation response transcription factor 2) overexpressors and pho2 (phosphate‐responsive mutant 2). Quantitative RT‐PCR showed that expression of OsSPX1 was strongly induced in OsPHR2 overexpression and pho2 mutant plants, indicating that OsSPX1 occurs downstream of OsPHR2 and PHO2. The expression of 10 genes associated with the phosphate‐starvation signal pathways was analyzed. Expression of OsPT2 (phosphate transporter 2) and OsPT8 was significantly induced in OsSPX1‐RNAi (OsSPX1‐Ri) plants, suggesting that over‐accumulation of Pi in OsSPX1‐Ri plants results from an increase in Pi transport. In contrast, overexpression of OsSPX1 suppressed the induction of expression by Pi starvation of all 10 phosphate starvation‐induced genes tested: IPS1 (induced by phosphate starvation 1), IPS2, OsPAP10 (purple acid phosphatase 10), OsSQD2 (sulfoquinovosyldiacylglycerol 2), miR399d and miR399j (microRNA 399), OsPT2, OsPT3, OsPT6 and OsPT8. This suggests that OsSPX1 acts via a negative feedback loop to optimize growth under phosphate‐limited conditions.

• Proteins possessing the SPX domain are found in several proteins involved in inorganic phosphate (Pi) transport and signalling in yeast and plants. Although the functions of several SPX‐domain protein subfamilies have recently been uncovered, the role of the SPX‐MFS subfamily is still unclear. • Using quantitative RT‐PCR analysis, we studied the regulation of SPX‐MFS gene expression by the central regulator, OsPHR2 and Pi starvation. The function of OsSPX‐MFS1 in Pi homeostasis was analysed using an OsSPX‐MFS1 mutant (mfs1) and osa‐miR827 overexpression line (miR827‐Oe). Finally, heterologous complementation of a yeast mutant impaired in Pi transporter was used to assess the capacity of OsSPX‐MFS1 to transport Pi. • Transcript analyses revealed that members of the SPX‐MFS family were mainly expressed in the shoots, with OsSPX‐MFS1 and OsSPX‐MFS3 being suppressed by Pi deficiency, while OsSPX‐MFS2 was induced. Mutation in OsSPX‐MFS1 (mfs1) and overexpression of the upstream miR827 (miR827‐Oe) plants impaired Pi homeostasis in the leaves. In addition, studies in yeast revealed that OsSPX‐MFS1 may be involved in Pi transport. • The results suggest that OsSPX‐MFS1 is a key player in maintaining Pi homeostasis in the leaves, potentially acting as a Pi transporter.

In the yeast Saccharomyces cerevisiae, a working model for nutrient homeostasis in eukaryotes, inorganic phosphate (Pi) homeostasis is regulated by the PHO pathway, a set of phosphate starvation induced genes, acting to optimize Pi uptake and utilization. Among these, a subset of proteins containing the SPX domain has been shown to be key regulators of Pi homeostasis. In this review, we summarize the recent progresses in elucidating the mechanisms controlling Pi homeostasis in yeast, focusing on the key roles of the SPX domain‐containing proteins in these processes, as well as describing the future challenges and opportunities in this fast‐moving field.