PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is prevalent in upland rice and enhances root growth and hastens low phosphate signaling in wheat.

PHOSPHORUS-STARVATION TOLERANCE 1 (OsPSTOL1) is a variably present gene that benefits crown root growth and phosphorus (P) sufficiency in rice (Oryza sativa). To explore the ecophysiological importance of this gene, we performed a biogeographic survey of landraces and cultivars, confirming that functional OsPSTOL1 alleles prevail in low nutrient and drought-prone rainfed ecosystems, whereas loss-of-function and absence haplotypes predominate in control-irrigated paddy varieties of east Asia. An evolutionary history analysis of OsPSTOL1 and related genes in cereal, determined it and other genes are kinase-only domain derivatives of membrane-associated receptor like kinases. Finally, to evaluate the potential value of this kinase of unknown function in another Gramineae, wheat (Triticum aestivum) lines overexpressing OsPSTOL1 were evaluated under field and controlled low P conditions. OsPSTOL1 enhances growth, crown root number, and overall root plasticity under low P in wheat. Survey of root and shoot crown transcriptomes at two developmental stages identifies transcription factors that are differentially regulated in OsPSTOL1 wheat that are similarly controlled by the gene in rice. In wheat, OsPSTOL1 alters the timing and amplitude of regulators of root development in dry soils and hastens induction of the core P-starvation response. OsPSTOL1 and related genes may aid more sustainable cultivation of cereal crops.

Transcriptional response of rice flag leaves to restricted external phosphorus supply during grain filling in rice cv. IR64.

Plant phosphorus (P) remobilisation during leaf senescence has fundamental implications for global P cycle fluxes. Hypothesising that genes involved in remobilisation of P from leaves during grain filling would show altered expression in response to P deprivation, we investigated gene expression in rice flag leaves at 8 days after anthesis (DAA) and 16 DAA in plants that received a continuous supply of P in the nutrient solution vs plants where P was omitted from the nutrient solution for 8 consecutive days prior to measurement. The transcriptional response to growth in the absence of P differed between the early stage (8 DAA) and the later stage (16 DAA) of grain filling. At 8 DAA, rice plants maintained production of energy substrates through upregulation of genes involved in photosynthesis. In contrast, at 16 DAA carbon substrates were produced by degradation of structural polysaccharides and over 50% of highly upregulated genes in P-deprived plants were associated with protein degradation and nitrogen/amino acid transport, suggesting withdrawal of P from the nutrient solution led to accelerated senescence. Genes involved in liberating inorganic P from the organic P compounds and vacuolar P transporters displayed differential expression depending on the stage of grain filling stage and timing of P withdrawal.

Remobilisation of phosphorus fractions in rice flag leaves during grain filling: Implications for photosynthesis and grain yields.

Phosphorus (P) is translocated from vegetative tissues to developing seeds during senescence in annual crop plants, but the impact of this P mobilisation on photosynthesis and plant performance is poorly understood. This study investigated rice (Oryza sativa L.) flag leaf photosynthesis and P remobilisation in a hydroponic study where P was either supplied until maturity or withdrawn permanently from the nutrient solution at anthesis, 8 days after anthesis (DAA) or 16 DAA. Prior to anthesis, plants received either the minimum level of P in nutrient solution required to achieve maximum grain yield ('adequate P treatment'), or received luxury levels of P in the nutrient solution ('luxury P treatment'). Flag leaf photosynthesis was impaired at 16 DAA when P was withdrawn at anthesis or 8 DAA under adequate P supply but only when P was withdrawn at anthesis under luxury P supply. Ultimately, reduced photosynthesis did not translate into grain yield reductions. There was some evidence plants remobilised less essential P pools (e.g. Pi) or replaceable P pools (e.g. phospholipid-P) prior to remobilisation of P in pools critical to leaf function such as nucleic acid-P and cytosolic Pi. Competition for P between vegetative tissues and developing grains can impair photosynthesis when P supply is withdrawn during early grain filling. A reduction in the P sink strength of grains by genetic manipulation may enable leaves to sustain high rates of photosynthesis until the later stages of grain filling.

Phosphorus remobilization from rice flag leaves during grain filling: an RNA-seq study.

The physiology and molecular regulation of phosphorus (P) remobilization from vegetative tissues to grains during grain filling is poorly understood, despite the pivotal role it plays in the global P cycle. To test the hypothesis that a subset of genes involved in the P starvation response are involved in remobilization of P from flag leaves to developing grains, we conducted an RNA-seq analysis of rice flag leaves during the preremobilization phase (6 DAA) and when the leaves were acting as a P source (15 DAA). Several genes that respond to phosphate starvation, including three purple acid phosphatases (OsPAP3, OsPAP9b and OsPAP10a), were significantly up-regulated at 15 DAA, consistent with a role in remobilization of P from flag leaves during grain filling. A number of genes that have not been implicated in the phosphate starvation response, OsPAP26, SPX-MFS1 (a putative P transporter) and SPX-MFS2, also showed expression profiles consistent with involvement in P remobilization from senescing flag leaves. Metabolic pathway analysis using the KEGG system suggested plastid membrane lipid synthesis is a critical process during the P remobilization phase. In particular, the up-regulation of OsPLDz2 and OsSQD2 at 15 DAA suggested phospholipids were being degraded and replaced by other lipids to enable continued cellular function while liberating P for export to developing grains. Three genes associated with RNA degradation that have not previously been implicated in the P starvation response also showed expression profiles consistent with a role in P mobilization from senescing flag leaves.

Improving phosphorus use efficiency: a complex trait with emerging opportunities.

Phosphorus (P) is one of the essential nutrients for plants, and is indispensable for plant growth and development. P deficiency severely limits crop yield, and regular fertilizer applications are required to obtain high yields and to prevent soil degradation. To access P from the soil, plants have evolved high- and low-affinity Pi transporters and the ability to induce root architectural changes to forage P. Also, adjustments of numerous cellular processes are triggered by the P starvation response, a tightly regulated process in plants. With the increasing demand for food as a result of a growing population, the demand for P fertilizer is steadily increasing. Given the high costs of fertilizers and in light of the fact that phosphate rock, the source of P fertilizer, is a finite natural resource, there is a need to enhance P fertilizer use efficiency in agricultural systems and to develop plants with enhanced Pi uptake and internal P-use efficiency (PUE). In this review we will provide an overview of continuing relevant research and highlight different approaches towards developing crops with enhanced PUE. In this context, we will summarize our current understanding of root responses to low phosphorus conditions and will emphasize the importance of combining PUE with tolerance of other stresses, such as aluminum toxicity. Of the many genes associated with Pi deficiency, this review will focus on those that hold promise or are already at an advanced stage of testing (OsPSTOL1, AVP1, PHO1 and OsPHT1;6). Finally, an update is provided on the progress made exploring alternative technologies, such as phosphite fertilizer.

A novel allele of the P-starvation tolerance gene OsPSTOL1 from African rice (Oryza glaberrima Steud) and its distribution in the genus Oryza.

KEY MESSAGE: We have developed allele-specific markers for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs, to improve their tolerance to P-deficient soil. The deficiency of phosphorus (P) in soil is a major problem in Sub-Saharan Africa due to general nutrient depletion and the presence of P-fixing soils. Developing rice cultivars with enhanced P efficiency would, therefore, represent a sustainable strategy to improve the livelihood of resource-poor farmers. Recently the Pup1 locus, a major QTL for tolerance to P deficiency in soil, was successfully narrowed-down to a major gene, the protein kinase OsPSTOL1 (P-starvation tolerance), which was found to be generally absent from modern irrigated rice varieties. Our target is to improve the tolerance of African mega-varieties to P deficiency through marker-assisted introgression of PSTOL1. As a first step, we have determined the Pup1 haplotype and surveyed the presence or absence of PSTOL1 and other genes of the Pup1 locus in African mega-varieties, NERICAs (New Rice for Africa) and their Oryza glaberrima parents. Here, we report the presence of a novel PSTOL1 allele in upland NERICAs that was inherited from the O. glaberrima parent CG14. This allele showed a 35 base-pair substitution when aligned to the Kasalath allele, but maintained a fully conserved kinase domain, and is present in most O. glaberrima accessions evaluated. In-silico and marker analysis indicated that many other genes of the Kasalath Pup1 locus were missing in the O. glaberrima genome, including the dirigent-like gene OsPupK20-2, which was shown to be downstream of PSTOL1. We have developed several allele-specific markers for the use for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs.

The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency.

As an essential macroelement for all living cells, phosphorus is indispensable in agricultural production systems. Natural phosphorus reserves are limited, and it is therefore important to develop phosphorus-efficient crops. A major quantitative trait locus for phosphorus-deficiency tolerance, Pup1, was identified in the traditional aus-type rice variety Kasalath about a decade ago. However, its functional mechanism remained elusive until the locus was sequenced, showing the presence of a Pup1-specific protein kinase gene, which we have named phosphorus-starvation tolerance 1 (PSTOL1). This gene is absent from the rice reference genome and other phosphorus-starvation-intolerant modern varieties. Here we show that overexpression of PSTOL1 in such varieties significantly enhances grain yield in phosphorus-deficient soil. Further analyses show that PSTOL1 acts as an enhancer of early root growth, thereby enabling plants to acquire more phosphorus and other nutrients. The absence of PSTOL1 and other genes-for example, the submergence-tolerance gene SUB1A-from modern rice varieties underlines the importance of conserving and exploring traditional germplasm. Introgression of this quantitative trait locus into locally adapted rice varieties in Asia and Africa is expected to considerably enhance productivity under low phosphorus conditions.

Developing rice with high yield under phosphorus deficiency: Pup1 sequence to application.

The major quantitative trait locus (QTL) Phosphorus uptake1 (Pup1) confers tolerance of phosphorus deficiency in soil and is currently one of the most promising QTLs for the development of tolerant rice (Oryza sativa) varieties. To facilitate targeted introgression of Pup1 into intolerant varieties, the gene models predicted in the Pup1 region in the donor variety Kasalath were used to develop gene-based molecular markers that are evenly distributed over the fine-mapped 278-kb QTL region. To validate the gene models and optimize the markers, gene expression analyses and partial allelic sequencing were conducted. The markers were tested in more than 80 diverse rice accessions revealing three main groups with different Pup1 allele constitution. Accessions with tolerant (group I) and intolerant (group III) Pup1 alleles were distinguished from genotypes with Kasalath alleles at some of the analyzed loci (partial Pup1; group II). A germplasm survey additionally confirmed earlier data showing that Pup1 is largely absent from irrigated rice varieties but conserved in varieties and breeding lines adapted to drought-prone environments. A core set of Pup1 markers has been defined, and sequence polymorphisms suitable for single-nucleotide polymorphism marker development for high-throughput genotyping were identified. Following a marker-assisted backcrossing approach, Pup1 was introgressed into two irrigated rice varieties and three Indonesian upland varieties. First phenotypic evaluations of the introgression lines suggest that Pup1 is effective in different genetic backgrounds and environments and that it has the potential to significantly enhance grain yield under field conditions.

Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1.

Marker-assisted breeding is a very useful tool for breeders but still lags behind its potential because information on the effect of quantitative trait loci (QTLs) in different genetic backgrounds and ideal molecular markers are unavailable. Here, we report on some first steps toward the validation and application of the major rice QTL Phosphate uptake 1 (Pup1) that confers tolerance of phosphorus (P) deficiency in rice (Oryza sativa L.). Based on the Pup1 genomic sequence of the tolerant donor variety Kasalath that recently became available, markers were designed that target (1) putative genes that are partially conserved in the Nipponbare reference genome and (2) Kasalath-specific genes that are located in a large insertion-deletion (INDEL) region that is absent in Nipponbare. Testing these markers in 159 diverse rice accessions confirmed their diagnostic value across genotypes and showed that Pup1 is present in more than 50% of rice accessions adapted to stress-prone environments, whereas it was detected in only about 10% of the analyzed irrigated/lowland varieties. Furthermore, the Pup1 locus was detected in more than 80% of the analyzed drought-tolerant rice breeding lines, suggesting that breeders are unknowingly selecting for Pup1. A hydroponics experiment revealed genotypic differences in the response to P deficiency between upland and irrigated varieties but confirmed that root elongation is independent of Pup1. Contrasting Pup1 near-isogenic lines (NILs) were subsequently grown in two different P-deficient soils and environments. Under the applied aerobic growth conditions, NILs with the Pup1 locus maintained significantly higher grain weight plant(-1) under P deprivation in comparison with intolerant sister lines without Pup1. Overall, the data provide evidence that Pup1 has the potential to improve yield in P-deficient and/or drought-prone environments and in diverse genetic backgrounds.

Comparative sequence analyses of the major quantitative trait locus phosphorus uptake 1 (Pup1) reveal a complex genetic structure.

The phosphorus uptake 1 (Pup1) locus was identified as a major quantitative trait locus (QTL) for tolerance of phosphorus deficiency in rice. Near-isogenic lines with the Pup1 region from tolerant donor parent Kasalath typically show threefold higher phosphorus uptake and grain yield in phosphorus-deficient field trials than the intolerant parent Nipponbare. In this study, we report the fine mapping of the Pup1 locus to the long arm of chromosome 12 (15.31-15.47 Mb). Genes in the region were initially identified on the basis of the Nipponbare reference genome, but did not reveal any obvious candidate genes related to phosphorus uptake. Kasalath BAC clones were therefore sequenced and revealed a 278-kbp sequence significantly different from the syntenic regions in Nipponbare (145 kb) and in the indica reference genome of 93-11 (742 kbp). Size differences are caused by large insertions or deletions (INDELs), and an exceptionally large number of retrotransposon and transposon-related elements (TEs) present in all three sequences (45%-54%). About 46 kb of the Kasalath sequence did not align with the entire Nipponbare genome, and only three Nipponbare genes (fatty acid alpha-dioxygenase, dirigent protein and aspartic proteinase) are highly conserved in Kasalath. Two Nipponbare genes (expressed proteins) might have evolved by at least three TE integrations in an ancestor gene that is still present in Kasalath. Several predicted Kasalath genes are novel or unknown genes that are mainly located within INDEL regions. Our results highlight the importance of sequencing QTL regions in the respective donor parent, as important genes might not be present in the current reference genomes.

Assessing hybrid sterility in Oryza glaberrima x O. sativa hybrid progenies by PCR marker analysis and crossing with wide compatibility varieties.

Interspecific crossing of the African indigenous rice Oryza glaberrima with Oryza sativa cultivars is hindered by crossing barriers causing 100% spikelet sterility in F(1) hybrids. Since hybrids are partially female fertile, fertility can be restored by back crossing (BC) to a recurrent male parent. Distinct genetic models on spikelet sterility have been developed predicting, e.g., the existence of a gamete eliminator and/or a pollen killer. Linkage of sterility to the waxy starch synthase gene and the chromogen gene C, both located on chromosome 6, have been demonstrated. We selected a segregating BC(2)F(3) population of semi-sterile O. glaberrima x O. sativa indica hybrid progenies for analyses with PCR markers located at the respective chromosome-6 region. These analyses revealed that semi-sterile plants were heterozygous for a marker (OSR25) located in the waxy promoter, whereas fertile progenies were homozygous for the O. glaberrima allele. Adjacent markers showed no linkage to spikelet sterility. Semi-sterility of hybrid progenies was maintained at least until the F(4) progeny generation, suggesting the existence of a pollen killer in this plant material. Monitoring of reproductive plant development showed that spikelet sterility was at least partially due to an arrest of pollen development at the microspore stage. In order to address the question whether genes responsible for F(1) sterility in intraspecific hybrids ( O. sativa indica x japonica) also cause spikelet sterility in interspecific hybrids, crossings with wide compatibility varieties (WCV) were performed. WCV accessions possess "neutral" S-loci ( S(n)) improving fertility in intraspecific hybrids. This experiment showed that the tested S(n)-loci had no fertility restoring effect in F(1) interspecific hybrids. Pollen development was completely arrested at the microspore stage and grains were never obtained after selfing. This suggests that distinct or additional S-loci are responsible for sterility of O. glaberrima x O. sativa hybrids.