QTL mapping for early root and shoot vigor of upland rice (Oryza sativa L.) under P deficient field conditions in Japan and Madagascar.

Upland rice production is limited by the low phosphorus (P) availability of many highly weathered tropical soils and P deficiency is likely to become increasingly limiting in future drier climates because P mobility decreases sharply with soil moisture. Good seedling root development will be crucial to cope with the combined effects of low P and water availability. Upland rice genebank accession DJ123 was used as a donor for P efficiency and root vigor traits in a cross with inefficient local variety Nerica4 and a set of backcross lines were used to characterize the seedling stage response of upland rice to low P availability and to identify associated QTL in field trials in Japan and Madagascar. Ten QTL were detected for crown root number, root, shoot and total dry weight per plant in a highly P deficient field in Japan using the BC1F3 generation. Of these, qPef9 on chromosome 9 affected multiple traits, increasing root number, root weight and total biomass, whereas a neighboring QTL on chromosome 9 (qPef9-2) increased shoot biomass. Field trials with derived BC1F5 lines in a low-P field in Madagascar confirmed a highly influential region on chromosome 9. However, qPef9-2 appeared more influential than qPef9, as the shoot and root biomass contrast between lines carrying DJ123 or Nerica4 alleles at qPef9-2 was +23.8% and +13.5% compared to +19.2% and +14.4% at qPef9. This advantage increased further during the growing season, leading to 46% higher shoot biomass at the late vegetative stage. Results suggest an introgression between 8.0 and 12.9 Mb on chromosome 9 from P efficient donor DJ123 can improve plant performance under P-limited conditions. The QTL identified here have practical relevance because they were confirmed in the target genetic background of the local variety Nerica4 and can therefore be applied directly to improve its performance.

Comparative transcriptome analysis reveals a rapid response to phosphorus deficiency in a phosphorus-efficient rice genotype.

Phosphorus (P) is an essential plant nutrient. Most rice growing lands lack adequate P, requiring multiple P fertiliser applications to obtain expected yields. However, P fertiliser is environmentally damaging, and already unaffordable to the marginal farmers. This warrants developing P-efficient rice varieties that require less P to produce the expected yield. However, genetic factors underlying P-use efficiency (PUE) in rice remain elusive. Here, we conducted comparative transcriptome analysis using two rice varieties with contrasting PUE; a P-efficient landrace DJ123 and a P-inefficient modern cultivar IR64. We aimed to understand the transcriptomic responses in DJ123 that allow it to achieve a high PUE under low P conditions. Our results showed that both DJ123 and IR64 had replete tissue P concentrations after 48 h of P deprivation. Yet, DJ123 strongly responded to the external low P availability by inducing P starvation-inducible genes that included SPX2, PHO1, PAPs and SQDs, while these genes were not significantly induced in IR64. We envisage that the ability of DJ123 to rapidly respond to low P conditions might be the key to its high PUE. Our findings lay a valuable foundation in elucidating PUE mechanism in rice, thus will potentially contribute to developing P-efficient modern rice variety.

Genomic prediction of zinc-biofortification potential in rice gene bank accessions.

KEY MESSAGE: A genomic prediction model successfully predicted grain Zn concentrations in 3000 gene bank accessions and this was verified experimentally with selected potential donors having high on-farm grain-Zn in Madagascar. Increasing zinc (Zn) concentrations in edible parts of food crops, an approach termed Zn-biofortification, is a global breeding objective to alleviate micro-nutrient malnutrition. In particular, infants in countries like Madagascar are at risk of Zn deficiency because their dominant food source, rice, contains insufficient Zn. Biofortified rice varieties with increased grain Zn concentrations would offer a solution and our objective is to explore the genotypic variation present among rice gene bank accessions and to possibly identify underlying genetic factors through genomic prediction and genome-wide association studies (GWAS). A training set of 253 rice accessions was grown at two field sites in Madagascar to determine grain Zn concentrations and grain yield. A multi-locus GWAS analysis identified eight loci. Among these, QTN_11.3 had the largest effect and a rare allele increased grain Zn concentrations by 15%. A genomic prediction model was developed from the above training set to predict Zn concentrations of 3000 sequenced rice accessions. Predicted concentrations ranged from 17.1 to 40.2 ppm with a prediction accuracy of 0.51. An independent confirmation with 61 gene bank seed samples provided high correlations (r = 0.74) between measured and predicted values. Accessions from the aus sub-species had the highest predicted grain Zn concentrations and these were confirmed in additional field experiments, with one potential donor having more than twice the grain Zn compared to a local check variety. We conclude utilizing donors from the aus sub-species and employing genomic selection during the breeding process is the most promising approach to raise grain Zn concentrations in rice.

Phenotyping of a rice (Oryza sativa L.) association panel identifies loci associated with tolerance to low soil fertility on smallholder farm conditions in Madagascar.

Rice (Oryza sativa L.) is a staple food of Madagascar, where per capita rice consumption is among the highest worldwide. Rice in Madagascar is mainly grown on smallholder farms on soils with low fertility and in the absence of external inputs such as mineral fertilizers. Consequently, rice productivity remains low and the gap between rice production and consumption is widening at the national level. This study evaluates genetic resources imported from the IRRI rice gene bank to identify potential donors and loci associated with low soil fertility tolerance (LFT) that could be utilized in improving rice yield under local cultivation conditions. Accessions were grown on-farm without fertilizer inputs in the central highlands of Madagascar. A Genome-wide association study (GWAS) identified quantitative trait loci (QTL) for total panicle weight per plant, straw weight, total plant biomass, heading date and plant height. We detected loci at locations of known major genes for heading date (hd1) and plant height (sd1), confirming the validity of GWAS procedures. Two QTLs for total panicle weight were detected on chromosomes 5 (qLFT5) and 11 (qLFT11) and superior panicle weight was conferred by minor alleles. Further phenotyping under P and N deficiency suggested qLFT11 to be related to preferential resource allocation to root growth under nutrient deficiency. A donor (IRIS 313-11949) carrying both minor advantageous alleles was identified and crossed to a local variety (X265) lacking these alleles to initiate variety development through a combination of marker-assisted selection with selection on-farm in the target environment rather than on-station as typically practiced.

Leaf phosphorus fractionation in rice to understand internal phosphorus-use efficiency.

BACKGROUND AND AIMS: Phosphorus (P) availability is often limiting for rice (Oryza sativa) production. Improving internal P-use efficiency (PUE) is crucial to sustainable food production, particularly in low-input systems. A critical aspect of PUE in plants, and one that remains poorly understood, is the investment of leaf P in different chemical P fractions (nucleic acid-P, lipid-P, inorganic-P, metabolite-P and residual-P). The overarching objective of this study was to understand how these key P fractions influence PUE. METHODS: Three high-PUE and two low-PUE rice genotypes were grown in hydroponics with contrasting P supplies. We measured PUE, total P, P fractions, photosynthesis and biomass. KEY RESULTS: Low investment in lipid-P was strongly associated with increased photosynthetic PUE (PPUE), achieved by reducing total leaf P concentration while maintaining rapid photosynthetic rates. All low-P plants exhibited a low investment in inorganic-P and lipid-P, but not nucleic acid-P. In addition, whole-plant PUE was strongly associated with reduced total P concentration, increased biomass and increased preferential allocation of resources to the youngest mature leaves. CONCLUSIONS: Lipid remodelling has been shown in rice before, but we show for the first time that reduced lipid-P investment improves PUE in rice without reducing photosynthesis. This presents a novel pathway for increasing PUE by targeting varieties with reduced lipid-P investment. This will benefit rice production in low-P soils and in areas where fertilizer use is limited, improving global food security by reducing P fertilizer demands and food production costs.

From gene banks to farmer's fields: using genomic selection to identify donors for a breeding program in rice to close the yield gap on smallholder farms.

KEY MESSAGE: Despite phenotyping the training set under unfavorable conditions on smallholder farms in Madagascar, we were able to successfully apply genomic prediction to select donors among gene bank accessions. Poor soil fertility and low fertilizer application rates are main reasons for the large yield gap observed for rice produced in sub-Saharan Africa. Traditional varieties that are preserved in gene banks were shown to possess traits and alleles that would improve the performance of modern variety under such low-input conditions. How to accelerate the utilization of gene bank resources in crop improvement is an unresolved question and here our objective was to test whether genomic prediction could aid in the selection of promising donors. A subset of the 3,024 sequenced accessions from the IRRI rice gene bank was phenotyped for yield and agronomic traits for two years in unfertilized farmers' fields in Madagascar, and based on these data, a genomic prediction model was developed. This model was applied to predict the performance of the entire set of 3024 accessions, and the top predicted performers were sent to Madagascar for confirmatory trials. The prediction accuracies ranged from 0.10 to 0.30 for grain yield, from 0.25 to 0.63 for straw biomass, to 0.71 for heading date. Two accessions have subsequently been utilized as donors in rice breeding programs in Madagascar. Despite having conducted phenotypic evaluations under challenging conditions on smallholder farms, our results are encouraging as the prediction accuracy realized in on-farm experiments was in the range of accuracies achieved in on-station studies. Thus, we could provide clear empirical evidence on the value of genomic selection in identifying suitable genetic resources for crop improvement, if genotypic data are available.

Identification of Loci Through Genome-Wide Association Studies to Improve Tolerance to Sulfur Deficiency in Rice.

Sulfur (S) is an essential nutrient for plant growth and development; however, S supply for crop production is decreasing due to reduced inputs from atmospheric deposition and reduced application of S-containing fertilizers. Sulfur deficiency in soil is therefore becoming a widespread cause of reduced grain yield and quality in rice (Oryza sativa L). We therefore assessed the genotypic variation for tolerance to S deficiency in rice and identified loci associated with improved tolerance. Plants were grown in nutrient solution with either low (0.01 mM) or high (1.0 mM) supply of S. Plants grown under low-S treatment showed a reduction in total biomass, mainly due to a marked reduction in shoot biomass, while root biomass and root-to-shoot ratio increased, relative to plants under high-S treatment. Genome-wide association studies (GWAS) identified loci associated with root length (qSUE2-3, qSUE4, and qSUE9), and root (qSUE1, qSUE2-1, and qSUE3-1 and qSUE3-2) or total dry matter (qSUE2, qSUE3-1, and qSUE11). Candidate genes identified at associated loci coded for enzymes involved in secondary S metabolic pathways (sulfotransferases), wherein the sulfated compounds play several roles in plant responses to abiotic stress; cell wall metabolism including wall loosening and modification (carbohydrate hydrolases: beta-glucosidase and beta-gluconase) important for root growth; and cell detoxification (glutathione S-transferase). This study confirmed the existence of genetic variation conferring tolerance to S deficiency among traditional aus rice varieties. The advantageous haplotypes identified could be exploited through marker assisted breeding to improve tolerance to S-deficiency in modern cultivars in order to achieve sustainable crop production and food security.

Genome-wide association and gene validation studies for early root vigour to improve direct seeding of rice.

Elucidation of the genetic control of rice seedling vigour is now paramount with global shifts towards direct seeding of rice and the consequent demand for early vigour traits in breeding programmes. In a genome-wide association study using an indica-predominant diversity panel, we identified quantitative trait loci (QTLs) for root length and root number in rice seedlings. Among the identified QTLs, one QTL for lateral root number on chromosome 11, qTIPS-11, was associated with a 32.4% increase in lateral root number. The locus was validated in independent backgrounds, and a predicted glycosyl hydrolase, TIPS-11-9, was identified as the causal gene for observed phenotypic differences. TIPS-11-9 was differentially expressed in emerging lateral roots of contrasting qTIPS-11 haplotypes, which was likely due to differences in cis-regulatory elements and auxin responsiveness. Abolishment of Tips-11-9 function through T-DNA insertion in a qTIPS-11-positive background resulted in a reduction of lateral root number, which negatively affected biomass accumulation, particularly under phosphorous-limiting conditions. Marker-assisted introgression of qTIPS-11 into modern indica varieties will aid in the generation of varieties adapted to direct seeding and thus facilitate the adoption of direct seeding practices in tropical Asia.

Phosphorus uptake commences at the earliest stages of seedling development in rice.

Seed phosphorus (P) reserves are essential for seedling development; however, we hypothesise that the quantity of P in seeds will lose importance in cultivars that rapidly acquire it via their roots. Our objective in this study was therefore to investigate the onset of seedling P uptake in rice (Oryza sativa). This was addressed through 33P-labelled supply and through measuring P depletion in combination with the detection of P transporter activity in the root tissue of three rice cultivars during early development. 33P supplied to roots 4 d after germination (DAG) was detected in shoots 2 d later, indicating that P was taken up and translocated to shoots during early seedling development. Measurements of P depletion from the growth medium indicated that uptake occurred even at 2 DAG when roots were only 3 cm long. By day 3, P depletion was rapid and P transporter activity was detected in roots, regardless of the levels of seed P reserves present. We conclude that P uptake commences at the earliest stages of seedling development in rice, that the amount taken up will be limited by root size, and that genotypes with more rapid root development should more rapidly complement seed-P reserves by root uptake.

The role of root size versus root efficiency in phosphorus acquisition in rice.

In rice, genotypic differences in phosphorus (P) uptake from P-deficient soils are generally proportional to differences in root biomass or surface area (RSA). It is not known to what extent genotypic variation for root efficiency (RE) exists or contributes to P uptake. We evaluated 196 rice accessions under P deficiency and detected wide variation for root biomass which was significantly associated with plant performance. However, at a given root size, up to 3-fold variation in total biomass existed, indicating that genotypes differed in how efficiently their root system acquired P to support overall plant growth. This was subsequently confirmed, identifying a traditional genotype, DJ123, with 2.5-fold higher RE (32.5 µg P cm(-2) RSA) compared with the popular modern cultivar IR64. A P depletion experiment indicated that RE could not be explained by P uptake kinetics since even IR64 depleted P to <20nM. A genome-wide association study identified loci associated with RE, and in most cases the more common marker type improved RE. This may indicate that modern rice cultivars lost the ability for efficient P uptake, possibly because they were selected under highly fertile conditions. One association detected on chromosome 11 that was present in a small group of seven accessions (including DJ123) improved RE above the level already present in many traditional rice accessions. This subspecies is known to harbor genes enhancing stress tolerance, and DJ123 may thus serve as a donor of RE traits and genes that modern cultivars seem to have lost.

Unmasking Novel Loci for Internal Phosphorus Utilization Efficiency in Rice Germplasm through Genome-Wide Association Analysis.

Depletion of non-renewable rock phosphate reserves and phosphorus (P) fertilizer price increases has renewed interest in breeding P-efficient varieties. Internal P utilization efficiency (PUE) is of prime interest because there has been no progress to date in breeding for high PUE. We characterized the genotypic variation for PUE present within the rice gene pool by using a hydroponic system that assured equal plant P uptake, followed by mapping of loci controlling PUE via Genome-Wide Association Studies (GWAS). Loci associated with PUE were mapped on chromosomes 1, 4, 11 and 12. The highest PUE was associated with a minor indica-specific haplotype on chromosome 1 and a rare aus-specific haplotype on chromosome 11. Comparative variant and expression analysis for genes contained within the chromosome 1 haplotype identified high priority candidate genes. Differences in coding regions and expression patterns between genotypes of contrasting haplotypes, suggested functional alterations for two predicted nucleic acid-interacting proteins that are likely causative for the observed differences in PUE. The loci reported here are the first identified for PUE in any crop that is not confounded by differential P uptake among genotypes. Importantly, modern rice varieties lacked haplotypes associated with superior PUE, and would thus benefit from targeted introgressions of these loci from traditional donors to improve plant growth in phosphorus-limited cropping systems.

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.

Root metabolic response of rice (Oryza sativa L.) genotypes with contrasting tolerance to zinc deficiency and bicarbonate excess.

Plants are routinely subjected to multiple environmental stresses that constrain growth. Zinc (Zn) deficiency and high bicarbonate are two examples that co-occur in many soils used for rice production. Here, the utility of metabolomics in diagnosing the effect of each stress alone and in combination on rice root function is demonstrated, with potential stress tolerance indicators identified through the use of contrasting genotypes. Responses to the dual stress of combined Zn deficiency and bicarbonate excess included greater root solute leakage, reduced dry matter production, lower monosaccharide accumulation and increased concentrations of hydrogen peroxide, phenolics, peroxidase and N-rich metabolites in roots. Both hydrogen peroxide concentration and root solute leakage were correlated with higher levels of citrate, allantoin and stigmasterol. Zn stress resulted in lower levels of the tricarboxylic acid (TCA) cycle intermediate succinate and the aromatic amino acid tyrosine. Bicarbonate stress reduced shoot iron (Fe) concentrations, which was reflected by lower Fe-dependent ascorbate peroxidase activity. Bicarbonate stress also favoured the accumulation of the TCA cycle intermediates malate, fumarate and succinate, along with the non-polar amino acid tyrosine. Genotypic differentiation revealed constitutively higher levels of D-gluconate, 2-oxoglutarate and two unidentified compounds in the Zn-efficient line RIL46 than the Zn-inefficient cultivar IR74, suggesting a possible role for these metabolites in overcoming oxidative stress or improving metal re-distribution.

Leaf ascorbic acid level--is it really important for ozone tolerance in rice?

Leaf ascorbic acid (ASA) level is thought to be an important trait conferring stress tolerance in plants, but definite evidence regarding its effectiveness in the breeding of stress tolerant crops is lacking. Therefore, the stress response of a rice TOS17 insertion mutant (ND6172) for a GDP-D-mannose-3',5'-epimerase gene, which is involved in ASA biosynthesis, was tested. Two fumigation experiments were conducted, in which rice plants (Oryza sativa L.) were exposed to (i) high ozone for ten days at the tillering stage (100 ppb, 7 h day⁻¹); and (ii) to four different ozone concentrations ranging from charcoal filtered air to 2.5 times the ambient concentration for the entire growth season. The mutant ND6172 had around 20-30% lower ASA level than the wild-type (Nipponbare), and exhibited a moderately higher level of visible leaf symptoms due to ozone exposure. Differences in ASA level between ND6172 and Nipponbare led to differential responses of the glutathione level, and the activities of glutathione reductase, ascorbate peroxidase, and dehydroascorbate reductase. With season-long ozone fumigation, yields and yield components were not negatively affected at ambient ozone level in both genotypes, but showed stronger decreases in ND6172 at higher ozone levels, especially at 2.5 times the ambient level. Similarly, the mature straw of ND6172 exhibited a higher degree of lignification at the 2.5 times ambient ozone level. In conclusion, a difference in leaf ASA level of around 20-30% is relevant for ozone tolerance in rice at levels exceeding the current ambient ozone concentrations.

The Frustration with Utilization: Why Have Improvements in Internal Phosphorus Utilization Efficiency in Crops Remained so Elusive?

Despite the attention internal phosphorus utilization efficiency (PUE) of crops has received in the literature, little progress in breeding crop cultivars with high PUE has been made. Surprisingly few studies have specifically investigated PUE; instead, genotypic variation for PUE has been investigated in studies that concurrently assess phosphorus acquisition efficiency (PAE). We hypothesized that genotypic differences in PAE confound PUE rankings because genotypes with higher PAE suffer a lower degree of P stress, resulting in lower PUE. The hypothesis was tested by comparing soil-based screening to a modified technique whereby rice genotypes were grown in individual containers with a single dose of solution P, to eliminate differences in P uptake among genotypes. Genotypic differences in PUE were apparent in root and shoot tissue using the modified nutrient solution technique, but PUE rankings showed no correlation with those from traditional soil-based screening. We conclude that PUE in soil-based screening systems is unavoidably linked with genotypic PAE, resulting in PUE rankings confounded by differences in P uptake. Only screening techniques assuring equal P uptake are suitable for the exploitation of genotypic variation for PUE.

Revisiting the role of organic acids in the bicarbonate tolerance of zinc-efficient rice genotypes.

It has been hypothesised that enhanced organic acid release from the roots of zinc-efficient rice (Oryza sativa L.) genotypes plays a strong role in plant tolerance to both bicarbonate excess and Zn deficiency. To address several uncertainties in the literature surrounding the tolerance of rice to bicarbonate, we initially assessed the tolerance of six rice genotypes to bicarbonate stress under field conditions and in solution culture. The landrace Jalmagna and its recombinant inbred offspring, RIL46, consistently performed better in terms of maintenance of biomass and root length under high bicarbonate concentrations. In the hydroponic experiments, increased root malate (but not citrate) accumulation and efflux were responses to high solution bicarbonate in the short-term (12h) in all genotypes. Although both citrate and malate accumulation and efflux increased after long-term exposure (10 days) to high bicarbonate and Zn deficiency, it coincided with amino acid leakage from the roots. Partial least-squares regression showed that this leakage consistently ranked highly as an indicator of poor plant health under all stress conditions, whereas specific malate efflux (the ratio of malate to amino acid efflux) was an important predictor of good plant health. The root leakage of Zn-inefficient genotypes under bicarbonate and dual stress (bicarbonate with low Zn) was typically higher than in Zn-efficient genotypes, and coincided with higher peroxide concentrations, suggesting that bicarbonate tolerance is related to the ability of Zn-efficient genotypes to overcome oxidative stress, maintain root membrane integrity and minimise root leakage.

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity.

*Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. *A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low [Zn](ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. *There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low [Zn](ext), correlating with altered expression of root-specific auxin-responsive genes. *Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low [Zn](ext); these traits are potential breeding targets.

Nitrification inhibition activity, a novel trait in root exudates of rice.

BACKGROUND AND AIMS: Nitrification is an important process in soil--plant systems for providing plant-available nitrate (NO(3) (-)). However, NO(3) (-) is less stable in soils compared with ammonium (NH(4) (+)) and is more easily lost through leaching, runoff or denitrification. This study tested whether biological nitrification inhibition (BNI) activity is present in the root exudates of rice (Oryza sativa) and also the extent of variation between different genotypes. METHODOLOGY: The BNI activity of root exudates was estimated by a bioluminescence assay using a recombinant Nitrosomonas europaea strain. Afterwards, the effect of a single application of concentrated root exudates and that of exudates deposited in the rhizosphere soil was tested on BNI using soil incubation. Soil was added with (NH(4))(2)SO(4) and water to reach 60 % of the water-holding capacity and incubated at 30 °C for different periods. Amounts of NH(4) (+) and NO(3) (-) were determined using a continuous-flow auto-analyser. PRINCIPAL RESULTS: In an initial screening experiment, BNI activity in the exudates of 36 different rice genotypes was evaluated using a bioassay based on a recombinant Nitrosomonas strain. Significant genotypic variation was detected with the upland cultivar IAC25 demonstrating consistently high BNI activity, while modern lowland varieties like Nipponbare or IR64 exhibited lower activity. Subsequent experiments ruled out the possibility that BNI activity is simply due to non-specific (solute) leakage from roots. Soil incubation studies with concentrated root exudates of IAC25 showed significant reductions in NO(3) (-) formation. This effect was confirmed by detecting lower NO(3) (-) levels in incubation experiments using rhizosphere soil obtained from IAC25. CONCLUSIONS: Our results provide first evidence that root exudates of rice can reduce nitrification rates in soil. Having shown this for a model crop, rice, offers possibilities for further exploitation of this phenomenon through molecular and genetic tools.