These authors contributed equally to this work. SummaryArabidopsis 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.

a b s t r a c tIn 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.

Summary 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.

Purple acid phosphatases (PAPs) are a family of metallo-phosphoesterases involved in a variety of physiological functions, especially phosphate deficiency adaptations in plants. We identified 26 putative PAP genes by a genome-wide analysis of rice (Oryza sativa), 24 of which have isolated EST sequences in the dbEST database. Amino acid sequence analysis revealed that 25 of these genes possess sets of metal-ligating residues typical of known PAPs. Phylogenetic analysis classified the 26 rice and 29 Arabidopsis PAPs into three main groups and seven subgroups. We detected transcripts of 21 PAP genes in roots or leaves of rice seedlings. The expression levels of ten PAP genes were up-regulated by both phosphate deprivation and over-expression of the transcription factor OsPHR2. These PAP genes all contained one or two OsPHR2 binding elements in their promoter regions, implying that they are directly regulated by OsPHR2. Both acid phosphatase (AP) and surface secretory acid phosphatase (SAP) activity assays showed that the up-regulation of PAPs by Pi starvation, OsPHR2 over-expression, PHO2 knockout or OsSPX1 RNA interference led to an increase in AP and SAP activity in rice roots. This study reveals the potential for developing technologies for crop improvement in phosphorus use efficiency.

The objective of this study was to evaluate the effects of dietary forage sources on milk performance, rumen microbial protein synthesis, and N utilization in early lactation dairy cows. Twelve primiparous Chinese Holstein dairy cows (45 ± 6.0 DIM) were used in a 3 × 3 Latin square design. Diets were isonitrogenous and isocaloric, with a forage-to-concentrate ratio of 45:55 [dry matter (DM) basis] and contained similar concentrate mixtures. Different forage sources were then added (on a DM basis): 21% corn silage, 19% corn stover, and 5% alfalfa hay (CS); 19% corn silage, 21% Chinese wild rye hay and 5% alfalfa hay (CWR); or 19% corn silage, 9% Chinese wild rye hay, and 17% alfalfa hay (AH). Each period lasted for 21 d, with the first 14 d for an adaptation period. Dry matter intake was not affected by the source of dietary forage. Milk yield was higher for cows fed AH than those fed CS, with an intermediate value for CWR. Milk protein content was higher in the cows fed AH compared with CWR (3.02 vs. 2.92%), with CS (2.95%) at an intermediate position. The contents of milk fat and lactose were not different among the treatments. However, milk efficiency (milk yield/DM intake) was higher for cows fed AH than those fed CS, with those fed CWR intermediate. Cows fed AH had higher microbial protein yield and metabolizable protein than those fed CS or CWR. The concentrations of urea N in the urine, blood, and milk were decreased for cows fed AH, indicating an increased N conversion. The results indicated that corn stover could replace Chinese wild rye grass in the diets for lactating cows and that a high proportion of alfalfa hay in the diet is beneficial for milk protein production by increasing microbial protein yield. This can be attributed to the improving the supply of rumen-available energy.