Matthias Wissuwa

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.

Genetic and genomic approaches to develop rice germplasm for problem soils

Soils that contain toxic amounts of minerals or are deficient in essential plant nutrients are widespread globally and seriously constrain rice production. New methods are necessary to incorporate the complex adaptive traits associated with tolerance of these abiotic stresses, while simultaneously retaining the high yield potential of rice varieties when conditions are favorable. Significant progress in the genetic characterization of stress response pathways and recent advances in genomics have provided powerful tools for in-depth dissection of tolerance mechanisms. Additionally, tolerance of most of these abiotic stresses in rice is controlled by a few QTLs with large effects despite the intricacy of the numerous traits involved. Genetic dissection of these QTLs and their incorporation into high-yielding varieties will significantly enhance and stabilize rice productivity in these problem soils. Current efforts at IRRI and in rice breeding programs worldwide are seeking to explore diverse germplasm collections and genetically dissect the causal mechanisms of tolerance to facilitate their use in breeding. This review focuses on salinity and P and Zn deficiency as the major problems encountered in rice soils, and examines current understanding of the mechanisms involved and efforts toward germplasm improvement.

Mapping of QTLs for phosphorus-deficiency tolerance in rice (Oryza sativa L.)

Abstract Phosphorus (P) deficiency of soils is a major yield-limiting factor in rice production. Increasing the P-deficiency tolerance of rice cultivars may represent a more cost-effective solution than relying on fertilizer application. The objective of this study was to identify putative QTLs for P-deficiency tolerance in rice, using 98 backcross inbred lines derived from a japonica×indica cross and genotyped at 245 RFLP marker loci. Lines were grown on P-deficient soil and P uptake, internal P-use efficiency, dry weight, and tiller number were determined. Three QTLs were identified for dry weight and four QTLs for P uptake, together explaining 45.4% and 54.5% of the variation for the respective traits. Peaks for both traits were in good agreement which was to be expected considering the tight correlation of r=0.96 between dry weight and P uptake. For both traits the QTL linked to marker C443 on chromosome 12 had a major effect. Two of the three QTLs detected for internal P-use efficiency, including the major one on chromosome 12, coincided with QTLs for P uptake; however, whereas indica alleles increased P uptake they reduced P-use efficiency. We concluded that this was not due to the tight linkage of two genes in repulsion but rather due to an indirect effect of P uptake on P-use efficiency. Most lines with high use efficiency were characterized by very low P uptake and dry weight and apparently experienced extreme P-deficiency stress. Their higher P-use efficiency was thus the result of highly sub-optimal tissue-P concentrations and did not represent a positive adaptation to low P availability. The number of tillers produced under P deficiency is viewed as an indirect indicator of P-deficiency tolerance in rice. In addition to the major QTL on chromosome 12 already identified for all other traits, two QTLs on chromosome 4 and 12 were identified for tiller number. Their position, however, coincided with QTLs for tiller number reported elsewhere under P-sufficient conditions and therefore appear to be not related to P-deficiency tolerance. In this study P-deficiency tolerance was mainly caused by differences in P uptake and not in P-use efficiency. Using a trait indirectly related to P-deficiency tolerance such as tiller number, we detected a major QTL but none of the minor QTLs detected for P uptake or dry weight.

Novel approaches in plant breeding for rhizosphere-related traits.

Selection of modern varieties has typically been performed in standardized, high fertility systems with a primary focus on yield. This could have contributed to the loss of plant genes associated with efficient nutrient acquisition strategies and adaptation to soil-related biotic and abiotic stresses if such adaptive strategies incurred a cost to the plant that compromised yield. Furthermore, beneficial interactions between plants and associated soil organisms may have been made obsolete by the provision of nutrients in high quantity and in readily plant available forms. A review of evidence from studies comparing older traditional varieties to modern high yielding varieties indeed showed that this has been the case. Given the necessity to use scarce and increasingly costly fertilizer inputs more efficiently while also raising productivity on poorer soils, it will be crucial to reintroduce desirable rhizosphere-related traits into elite cultivars. Traits that offer possibilities for improving nutrient acquisition capacity, plant–microbe interactions and tolerance to abiotic and biotic soil stresses in modern varieties were reviewed. Despite the considerable effort devoted to the identification of suitable donors and of genetic factors associated with these beneficial traits, progress in developing improved varieties has been slow and has so far largely been confined to modifications of traditional breeding procedures. Modern molecular tools have only very recently started to play a rather small role. The few successful cases reviewed in this paper have shown that novel breeding approaches using molecular tools do work in principle. When successful, they involved close collaboration between breeders and scientists conducting basic research, and confirmation of phenotypes in field tests as a ‘reality check’. We concluded that for novel molecular approaches to make a significant contribution to breeding for rhizosphere related traits it will be essential to narrow the gap between basic sciences and applied breeding through more interdisciplinary research that addresses rather than avoids the complexity of plant–soil interactions.

How Do Plants Achieve Tolerance to Phosphorus Deficiency? Small Causes with Big Effects

Genotypic differences in phosphorus (P) uptake from P-deficient soils may be due to higher root growth or higher external root efficiency (micrograms of P taken up per square centimeter of root surface area). Both factors are highly interrelated because any additional P provided by externally efficient roots will also stimulate root growth. It will be necessary to separate both factors to identify a primary mechanism to formulate hypotheses on pathways and genes causing genotypic differences in P uptake. For this purpose, a plant growth model was developed for rice (Oryza sativa) grown under highly P-deficient conditions. Model simulations showed that small changes in root growth-related parameters had big effects on P uptake. Increasing root fineness or the internal efficiency for root dry matter production (dry matter accumulated per unit P distributed to roots) by 22% was sufficient to increase P uptake by a factor of three. That same effect could be achieved by a 33% increase in external root efficiency. However, the direct effect of increasing external root efficiency accounted for little over 10% of the 3-fold increase in P uptake. The remaining 90% was due to enhanced root growth as a result of higher P uptake per unit root size. These results demonstrate that large genotypic differences in P uptake from a P-deficient soil can be caused by rather small changes in tolerance mechanisms. Such changes will be particularly difficult to detect for external efficiency because they are likely overshadowed by secondary root growth effects.

Effects of zinc deficiency on rice growth and genetic factors contributing to tolerance.

Zinc (Zn) deficiency is the most widespread micronutrient disorder in rice (Oryza sativa), but efforts to develop cultivars with improved tolerance have been hampered by insufficient understanding of genetic factors contributing to tolerance. The objective of this paper was to examine alternative evaluation methods and to identify the most informative traits that would provide realistic information for rice breeders and to map quantitative trait loci (QTLs) associated with tolerance. Screening experiments in low-Zn nutrient solution and in a Zn-deficient field did not produce similar tolerance rankings in a set of segregating lines, which suggested that rhizosphere effects were of greater importance for lowland rice than internal Zn efficiency. The most severe symptom in the field was high plant mortality. The occurrence of leaf bronzing, usually regarded as indicative of susceptibility, did not necessarily concur with high plant mortality, which implied that both were under independent genetic control. The QTL mapping experiment conducted in the field with a population derived from a cross of IR74 (intolerant) with Jalmagna (tolerant) largely confirmed this. Four QTLs associated with plant mortality were detected, and only one of those colocalized with one of the four QTLs detected for leaf bronzing. The two most influential QTLs for plant mortality were detected on chromosomes 2 and 12. They explained 16.6% and 24.2% of the variation, and alleles of the tolerant donor parent Jalmagna reduced mortality by 16.6% and 14.8%, respectively. QTLs for plant mortality acted in a purely additive manner, whereas digenic epistatic interactions were important for leaf bronzing.

Organic Anion Exudation by Lowland Rice (Oryza sativa L.) at Zinc and Phosphorus Deficiency

The objectives of this paper were to determine (1) if lowland rice (Oryza sativa L.) plants respond similarly to low zinc (Zn) and phosphorus (P) availability by increased root exudation of low-molecular weight organic anions (LMWOAs) and (2) if genotypic variation in tolerance to low soil supply of either Zn or P is related to LMWOA exudation rates. Exudation of LMWOAs can increase bioavailability of both Zn and P to the plant, through partly similar chemical mechanisms. We used seven lowland rice genotypes and showed in two experiments that genotypes that grow relatively well on a soil with low Zn availability also grow well on a sparingly soluble Ca-phosphate (r = 0.80, P = 0.03). We measured exudation rates of LMWOAs on nutrient solution and found that both Zn and P deficiency induced significant increases. Among the LMWOAs detected oxalate was quantitatively the most important, but citrate is considered more effective in mobilizing Zn. Citrate exudation rates correlated with tolerance to low soil levels of Zn (P=0.05) and P (P = 0.07). In a low-Zn-field we found an increased biomass production at higher plant density, which is supportive for a concentration-dependent rhizosphere effect on Zn bioavailability such as LMWOA exudation. We, for the first time, showed that tolerance to low Zn availability is related to the capacity of a plant to exude LMWOAs and confirmed that exudation of LMWOAs must be regarded a multiple stress response.

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.

Further characterization of two QTLs that increase phosphorus uptake of rice ( Oryza sativa L.) under phosphorus deficiency

Four quantitative trait loci (QTLs) for P uptake were previously identified in a rice population that had been developed from a cross between the indica landrace Kasalath (high P uptake) with the japonica cultivar Nipponbare (low P uptake). For further studies, near isogenic lines (NILs) were developed for a major QTL linked to marker C443 on chromosome 12 and for a minor QTL linked to C498 on chromosome 6. On a highly P-deficient upland soil (aerobic conditions), NIL-C443 had three to four times the P uptake of Nipponbare, whereas the advantage of NIL-C498 was in the range of 60–90%. The superiority of NILs over Nipponbare vanished when grown in the same soil under anaerobic paddy conditions. All genotypes had high P uptake when P was supplied at a rate of 60 kg P ha−1, regardless of soil conditions. These results confirmed the presence of both QTLs and furthermore implied that QTLs affected absorption mechanisms that specifically increased P uptake in a P deficient upland soil.Additional experiments were conducted to investigate if the effect of QTLs is linked to an increase in root growth or due to more efficient P uptake per unit root size (higher root efficiency). Root size did not differ significantly between genotypes in the plus-P treatment. P deficiency, however, reduced the root surface area of Nipponbare by more than 80% whereas NIL-C443 maintained almost half of its non-stress root surface area. The low root growth of Nipponbare observed under P deficiency was probably the result of insufficient P uptake to sustain plant growth, including root growth. Genotypic differences in the ability to maintain root growth, therefore are likely caused by some mechanism that increases the efficiency of roots to access P forms not readily available. This however, only had an effect in aerobic soil. Potential mechanisms leading to higher P uptake of NILs are discussed.

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.