Plants have evolved a number of adaptive responses to cope with growth in conditions of limited phosphate (Pi) supply involving biochemical, metabolic, and developmental changes. We prepared an EMS-mutagenized M₂ population of an Arabidopsis thaliana transgenic line harboring a reporter gene specifically responsive to Pi starvation (AtIPS1∷GUS), and screened for mutants altered in Pi starvation regulation. One of the mutants,phr1 (phosphate starvation response 1), displayed reduced response of AtIPS1∷GUS to Pi starvation, and also had a broad range of Pi starvation responses impaired, including the responsiveness of various other Pi starvation-induced genes and metabolic responses, such as the increase in anthocyanin accumulation. PHR1 was positionally cloned and shown be related to the PHOSPHORUS STARVATION RESPONSE 1(PSR1) gene from Chlamydomonas reinhardtii. A GFP∷PHR1 protein fusion was localized in the nucleus independently of Pi status, as is the case for PSR1. PHR1 is expressed in Pi sufficient conditions and, in contrast to PSR1, is only weakly responsive to Pi starvation. PHR1, PSR1, and other members of the protein family share a MYB domain and a predicted coiled–coil (CC) domain, defining a subtype within the MYB superfamily, the MYB–CC family. Therefore, PHR1 was found to bind as a dimer to an imperfect palindromic sequence. PHR1-binding sequences are present in the promoter of Pi starvation-responsive structural genes, indicating that this protein acts downstream in the Pi starvation signaling pathway.

Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress. As sessile organisms, plants are often exposed to stress conditions, and have evolved adaptive responses to protect themselves from different types of stress. Some responses are stress type-specific whereas others are common to different stress types. Understanding how these responses are controlled is crucial for rational improvement of stress tolerance, a limiting factor in crop productivity. Here we examined the physiological and molecular responses to phosphate starvation and found that a single transcription factor family, represented by PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1), has a central role in the control of specific and shared phosphate starvation stress responses. In consonance with the importance of PHR1, we found that the PHR1-binding sequence, present in most PHR1 direct targets, is a crucial cis motif for Pi starvation responsiveness. An artificial promoter controlled by PHR1 recapitulates responsiveness to Pi starvation and to modulators of this response, qualifying PHR1 family members as central integrators in Pi starvation signalling. This central integrator system also controls most transcriptional repression responses to Pi starvation, indicating that they are an integral part of the adaptive response, and not a consequence of plant malfunction due to stress.

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. PHOSPHATE STARVATION RESPONSE 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling. Significance When P levels are low, plants activate an array of adaptive responses to increase efficient acquisition and use of phosphate (Pi), the form in which P is preferentially absorbed, and to protect themselves from Pi starvation stress. Considerable progress has been made recently in dissecting the plant Pi starvation signaling pathway. Nonetheless, little is known as to how Pi levels are perceived by plants. Here, we identify the nuclear protein SPX1 as a Pi-dependent inhibitor of DNA binding by PHOSPHATE STARVATION RESPONSE 1 (PHR1), a master regulator of Pi starvation responses. We show that the Pi dependence of SPX1 inhibition of PHR1 activity can be recreated in vitro using purified proteins, which indicates that the SPX1/PHR1 module links Pi sensing and signaling.

Arabidopsis COP1 is a constitutive repressor of photomorphogenesis that interacts with photomorphogenesis-promoting factors such as HY5 to promote their proteasome-mediated degradation. SPA1 is a repressor of phytochrome A-mediated responses to far-red light. Here we report that COP1 acts as part of a large protein complex and interacts with SPA1 in a light-dependent manner. We further demonstrate the E3 ubiquitin ligase activity of COP1 on HY5 in vitro and the alteration of that activity by SPA1. Thus, the COP1-SPA1 interaction defines a critical step in coordinating COP1-mediated ubiquitination and subsequent degradation of HY5 with PHYA signaling.

Summary: Anthocyanins are natural pigments that accumulate only in light‐grown and not in dark‐grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA‐105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light‐mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark‐grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post‐translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants.